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Adenovirus Methods and Protocols
Adenovirus Methods and Protocols
Stock
Terry W. Hermiston,
1. Introduction
An important step m the development of modern experimental virology was
the development of the plaque assay, first with bacteriophage, then with
eukaryotic viruses. In order to obtain quantitative, interpretable, and reproducible results, it is necessary to know how much virus is bemg used in the experrment. With adenovuuses, several approaches have generally been used to
quantitate vrrus stocks. First, virus particles are counted, e.g., in an electron
microscope (1,2). Another approach is to quantitate virion DNA by optical
absorbance (2). The problem with these approaches is that many adenovirus
particles are not infectious, perhaps because they have a defective complete
genome or they lack fiber or some other protein. The second approach is to
determine the number of plaque-forming units (PFU) per mL. Here, the analysis quantitates the number of vnions capable of a full infectious cycle. This
approach will be described in detail in this article.
Experimental reproducibility also requires that adenovirus stocks be prepared in a consistent manner. Such stocks are stable for years when stored
at -7OOC. One simple approach is to prepare a cytopathic effect (CPE) stock.
Here, an isolated plaque is picked and a small dish of permissive cells (e.g.,
A549) is infected. After approx 4-5 d, the cells in the monolayer will show
typical adenovirus CPE, i.e., their nuclei will become enlarged and they will
round up and detach from the dishes into individual floating cells as well as
grape-like clusters. These floating cells remain alive for some time (cell death
and the release of adenovnus from the cells begins at approx 3 d postinfection
for subgroup C adenoviruses). These cells are collected, adenovirus is released
by repeatedly freezing and thawing, and then it is used to infect a larger monoFrom
and Protocols
NJ
layer, such as a T-150 flask. After CPE appears, cells are collected and adenovirus is released by three rounds of freeze-thawing followed by sonication;
then the adenovirus is titered by plaque assay. These CPE stocks, which are
typically 1Os-1Oo PFU/mL, are adequate for exploratory studies. Large-scale
vnus stocks are usually prepared by banding the virus in CsCl equilibriumdensity gradients, and this procedure will be described. CsCl-banding yields
large quantities of high-titer (10 t PFU/mL) adenovirus stocks.
2. Materials
2.1. Cell-Culture
1 Dulbeccos modified Eagles medium (DMEM) from powdered medmm (20 L):
DMEM (with high glucose, with L-glutamine, with phenol red, without sodmm
pyruvate, without sodium bicarbonate; Gtbco-BRL, Gaithersburg, MD; JRH Btosciences [Lenexa, KS]) (2X 10 L), 74 g (3.7 g/L) of sodmm bicarbonate (tissueculture grade, Gibco-BRL or Sigma [St. Louis, MO]). Adjust the pH of the
solution to approx 6.9 with 1 N HCl or 1 N NaOH (pH will increase 0.3 to 0.4 U
with filtration). Add commercral penicillin-streptomycm
stock (1 mL/L; GibcoBRL). Medium is membrane-sterilized by posrtive or negative pressure (postttve
pressure IS preferable in mamtaming pH) through a 0 22-p filter (Mrllipore,
Bedford, MA; Coming [Coming, NY], Nalge [Rochester, NY])
2. MEM (Joklik-modified)
for suspension cultures (20 L) (see Note 1) mmimum
essential medium (S-MEM) (Jokbk-modified) (with L-glutamine, with 10X phosphate, without sodmm bicarbonate; Gibco-BRL or JRH Biosciences) (2X 10 L),
40 g sodium bicarbonate (2 g/L) Adjust pH to 6.9 with 1 N HCI or 1 N NaOH,
then add streptomycin-penicillin
stock (1 mL/L). Sterilize medmm by membrane
filtration (0.22~pm filter).
3. 2X DME for plaque assay overlays (5 L) (see Note 2): Combine 4.5 L ddHzO
(tissue-culture grade), 0.6 g penicillin G (sodium salt, 1670 U/mg), 1.O g streptomycm sulfate (787 U/mg), DME powder (1X 10 L) (with htgh glucose, with
L-glutamine, with phenol red, without sodium bicarbonate, without sodium pyruvate; Gibco-BRL). AdJust to 5 L. pH is not adjusted at the time of preparation
(sodium bicarbonate stock is added at the time of overlay preparation). Sterilize
by membrane filtration through a 0.22-u filter
4. Penicillin and streptomycin: penicillin/streptomycin
stock (1000X) contains
10,000 U/mL penicillin G sodium, and 10,000 U/mL streptomycin sulfate in
0.85% saline (Gtbco-BRL). Store frozen until use in preparation of DMEM and
Joklik-modified
MEM.
5. Phosphate-buffered saline (PBS): prepare Dulbeccos phosphate-buffered saline
(PBS) (without calcium chloride, without magnesium chloride; Sigma) with trssue-culture grade water and sterilize by filtration through a 0.22~~.un filter.
6. Ttypsin-EDTA stock solution (4 L): dissolve 4 g trypsin (1:250, DIFCO Laboratories, Detroit, MI), 2 g EDTA (disodium salt), 4 g dextrose, 20 mg phenol red
(sodium salt, Fisher, Pittsburgh, PA), 0.25 g penicillin G (sodium salt, 1670 U/mg,
&Cl-Banded
7.
8.
9.
10.
Adenovirus Stock
3. Method
3.1. Growth of Monolayer
3.1.1. KB Cells
and Suspension
Cultures
2. Split the culture each day to a cell density of 1.5-2.0 x lo5 cells per mL. Cells
typically double in 24 h. Add new medium to the cell suspensionand discard
excesscells.Replaceonly a portion of the medium becauseconditioned medium
appearsto have some beneficial effect on the growth of the cells (perhaps from
autocrine effects).
3.1.2. A549 Cells
A549 cells (CCL 185; American Type Culture Collection, Rockville, MD)
are grown in DMEM supplemented with glutamine and 10% FBS (HyClone,
Logan, UT; BioWhittaker [Walkersville, MD]; or Gibco-BRL).
1, For routine passage,removemedium from theplatesandaddtrypsin-EDTA (2 mL
for 100~mmdish or 5 mL for a 175-cm2flask).
2. When cells have rounded up (usually in 3-5 min), remove cells from the dish
by adding DMEM (10% FBS) with gentle pipettmg. Use of 10% FBS/DMEM
inhibits continued trypsm action and cells remain more intact with higher
viability.
3. Centrifuge cells at 600-1000 rpm (lOO-25Og) in a table-top centrifuge (e.g.,
BeckmanGS-6) to pellet cells
4. Removetrypsin/medium solution andresuspendcells in DMEM (10% FBS), and
plate at 1:5 to 1:20 dilutions relative to the original cell density.
5. Cells areusually passagedat 2- to 3-d intervals. Grow cells at 37C with 6% CO*
in humidified incubators in 100~nundishesor 175-cm*flasks. Cells should not
be allowed to becomevery heavy in routine culture or they will not have good
survival in plaque assays.
3.1.3. Large-Scale Adenovirus Preparation
Spinner KB cells are used for large scale production of adenovnuses.
Grow cells m mmimal essential medium, Joklik-modified (Gibco-BRL or
JRH Biosciences), a suspension medtum with reduced calcium and
increased levels of phosphate, with 5% horse serum (heat-inactivated at
56OC for 30 min to inactivate complement). For infection, it 1s typical to
use a 3-L volume of cells that have reached a density of 3-3.5 x lo5 cells/ml.
Reduce volume for infection to 1 L by centrifuging 2400 mL of suspension
culture to pellet cells, resuspend these cells in approx 400 mL of serumfree Joklik-modified MEM, and return cells to spinner. Infect cells with 520 PFU/cell with stock viruses (lower MO1 will result in fewer defective
particles). Virus is adsorbed with spinning at 37OC for 1 h; at the end of the
adsorption period, add 2 L of medium (with 5% horse serum) to the infection. Maintain infected cells in spinner flasks at 37C for 40-46 h; then
harvest. Given viruses may be incubated for more extended times if cell
lysis is not occurring (especially if the E3 gene for the adenovirus death
protein, previously named E3-11.6K, is absent).
Day 1:
1. Prepare 3 L KB spmner cells in Joklik-modified
2 x lo5 cells per mL.
Day 2:
2. Do cell count on hemacytometer to determine the cell number. This cell number
will be used to determine the volume of virus to use for infection.
3. Reduce the total cell volume to 1 L by centrifugation. Cells (2400 mL) are
pelleted in a table-top centritige (Beckman GS-6 centrifuge) in 750-mL Beckman
centrifuge tubes at 1000 rpm (250g) for 10 min. Because the centrifuge bottle
bottoms are flat, the rotor is not braked at the end of the spin.
4 Resuspend cells in 400 mL Joklik-modified
medium (serum-free) and return to
the spinner flask.
5. Add virus (5-20 PFWcell or use a portion of a CPE stock from a flask). If using
small volumes of banded vnus, it is best to dilute the virus in serum-free Joklikmodified MEM in a 50-mL centrifuge tube (Falcon, Coming) prior to addition to
the spinner. Adsorb 1 h at 37C with spinning.
Day 4:
6. Pellet infected cells in 750-mL Beckman centrifuge tubes in table-top centrifuge
(1000 rpm [25Og], 10 min, 4C). Do not use brake at end of centrifugation.
7. Remove medium. Resuspend cell pellets m a total volume of 150-200 mL of
cold PBS (4C) and transfer to a 250-mL conical centrifuge tube (Corning). Centrifuge at 1000 rpm (250g) at 4C for 10 min.
8. Repeat PBS wash and pelleting of cells twice.
9. Resuspend cell pellet in enough cold 10 mM Tris-HCl, pH 8.0 (4C) to give a
final volume of 24 mL.
10. Ahquot 8 mL into each of three sterile polypropylene snap-cap tubes (15 mL
size), wrap caps with parafilm, and freeze at -7OC or m ethanol/dry ice bath for
at least 1 h (processing can be left at this point for one or more d before completing the remainder of the protocol).
11. Thaw tubes m 37C water bath. Repeat these freeze-thaw steps two more times
and then place tubes on ice.
12. Disrupt cells by sonication on me in the cup of a Branson sonifier 250. Settings
are as follows: duty cycle on constant, output control on 9 (scale of l-lo), and
3-mm cycles. Repeat three times for each sample.
13. Transfer somcated material to sterile 50-mL flip-cap centrifuge tubes and centnmge
at 10,000 rpm (12,000g) for 10 min at 4C in a Beckman J2-HC centrifuge. Remove
supematant (which will contain released virions) and discard cell-debris pellet.
14. Determine the volume of supernatant and multiply by 0.5 1. The resulting number
will be the grams of CsCl to be added to the preparation. For example: 20 mL
of supernatant x 0.5 1 g of CsCl per mL equals 10.2 g of CsCl for addition to
the supernatant.
15. After mixing wtth CsCI, divide sample into two Ti50 quick-seal tubes.
16. Centrrfuge in Ti50 rotor at 35,000 (110,OOOg) rpm m Beckman ultracentrifuge at
4C for 16-20 h to band the virus
Day 5:
17. Stop the ultracentrifnge without using the brake.
18 The virus will appear as a white band that will be at approximately the middle of
the tube. Collect by syringe puncture at the bottom (puncture top of tube as well).
Band will visibly move down the tube. Collect the band region in a 15- or 50-mL
sterile centrifuge tube (Coming, Falcon) as it drips from the bottom Altematively, the virus band can be removed by side puncture of the tube at the level of
the virus band with a syringe and withdrawing the band wtth the syringe
19. Dilute virus 5- to lo-fold in Trts-saline-glycerol (TSG) (see Subheading 2.1.,
item 8); this will usually result m a stock which is 101o-lO PFU/mL when using
wild-type viruses.
20 Aliquot in l- to 3-mL volumes in sterile 6-mL snap-cap polypropylene tubes or
m cryovials and store at -70C unttl needed.
21. Determine titer of the vtrus by plaque assay on A549 cells (see Note 5).
of Adenovirus
Titers
1. One day prior to plaque assay, plate A549 cells at 2.0 x lo6 tells/60-mm dish
(Coming, Falcon).
2 On the day of the plaque assay, wash dishes of confluent A549 cells with 5 mL
serum-free DMEM for 30-60 mm prtor to addition of the diluted vn-us; remove
this wash medium immediately before the addition of the VIIUS dilutions.
3. Make serial dtlutions of vtrus in serum-free DMEM; perform dilutions within a
laminar flow hood. Dilute virus in sterile-dtsposable snap-cap polypropylene
tubes and vortex well after each dilution (5-10 s at an 8-9 setting on a I-10
scale). Typically for cesium-chloride-banded stocks, the initial two dilutions are
1: 1000 (10 pL mto 10 mL), followed by dilutions of 1:lO. Be sure to change
micropipet tips after each dilution; avoid contamination of the mrcropipet barrel
(use of barrier tips will help avoid contammatton). Care should also be taken to
avoid transfer of virus stock on the outside of the micropipet tip by avoiding
dipping the tip into the solutton, especially in expellmg the volume. For cesiumbanded stocks, the range of dilutions that are usually countable are 1W8-1 O-lo.
Place a volume of 0.5 mL of the appropriate dilution on confluent A549 cells
(each relevant dilution 1sassayed in triplicate).
Rock dishes to distribute medium over the monolayer at lo- to 15-mm intervals.
Incubate cells at 37C with 6% CO,.
Immediately prior to addition of overlay to the cell monolayers, mix the agar
stock with the remaining ingredients
At the end of the 1-h adsorption period, add 6 mL overlay (see Subheading 2.1., item
9) to the edge of the dish and rotate the dish to blend overlay wtth the medium used
for infection (the 0.5-n& volume of medium used for infection IS not removed).
4. Notes
1. Incubate test bottles of medium at 37C for 5 d to ensure sterility for each batch.
Sterility can also be tested on blood agar plates or in nutrient broth. Care should
be taken to cover the medium during preparation and to prepare medium in an
area not normally used for handling of virus (adenovirus vtrions can pass
through a 0.22-p filter). Bottles, 20-L container, and stir bar used for media
preparation should be dedicated for tissue-culture use and not mixed with chemical glassware.
2. 2X DME is usually ahquoted in 500~mL volumes and is stored at 4C; care should
be taken to avoid increases in pH (cell survival of monolayers m plaque assays is
significantly decreased m medium that has become basic or if the pH of the
overlay is too high initially).
3. Serum testing: sera (HyClone, BioWhittaker, Gibco-BRL) are purchased in large
lots to reduce experimental variation caused by differences m serum lots. Sera
are tested with relevant cell lines in two different assays.
a. To determine cloning efficiency of cells at low-cell density, plate 100-500
cells per tissue culture plate. After 10-12 d, fix clones in methanol (10 min at
-20C) and stain wtth Giemsa staining solution or with crystal violet. The
number, stze, and morphology of clones can then be compared for dtfferent
lots. Determination of cloning efficiency is of importance for experiments in
which small numbers of cells will be present on a tissue-culture dish (as m
production of stable transfectants or in limited dilution for selectton of clonalcell populations). One can also check the appearance of clones and make subjective judgments about the growth of the cells (flatness or overgrowth of the
Tollefson,
4.
5.
6.
7.
Hermisron,and
Wold
clones, size of the cells, cell uniforrmty, vacuoles, mitotic index, and relative
health of the cells).
b Cell-growth rates are calculated by plating lo5 cells per 60-mm tissue-culture
dish; cell counts are done at daily intervals to determine the kinetics of growth
for the different serum lots.
The FBS is not heat-inactivated (heat-inactivation will substantially decrease the
survival of A549 cells in plaque assays).
Verification of virus stocks. Virus preparations are tested periodically by Hirt
assay (see Chapter 2) or other assays (such as immunofluorescence or PCR) to
confirm the fidelity of the vnus preparations. It may be necessary to plaquepurify a stock periodically (see Chapter 2) to eliminate possible contaminants
(this is especially important for viruses that grow less efficiently than wild-type
virus or that are released from cells less efficiently during infection).
Plaque-assay consistency: Careful and consistent plaque assays will typically
result in less than twofold differences in determined titer of the same virus preparation in separate experiments. It is often preferable to plaque assay a large panel
of mutants that will be used in the same experiments simultaneously so that the
relative titers will be quite accurate.
Adjusting plaque assays for small-plaque morphologies (viruses that lack the
subgroup C adenovnus death protein gene and serotypes that produce small
plaques): It is important to have consistent PFU informatton in order to perform
infections with viruses m which comparisons are made between the phenotypes
of various virus mutants. The most direct method of generating infectrous titers 1s
by doing plaque assays for PFU. In the study of E3 mutants, we have determined
that given mutants have a small plaque morphology and therefore a number of
modifications
have been made in previous plaque-assay methodologies to
accommodate the requirements for these mutants. It is necessary for the cell
monolayers to remain viable for an extended time (approx 28-30 d) to see the
full extent of the plaque development. Plaque assays are done on A549 cells
(ATCC) that have a very good survival time under the overlay in plaque assays.
Cell survrval of A549 cells is also somewhat dependent on the type of tissueculture dish used
References
1. Pinteric, L. and Taylor, J. (1962) The lowered drop method for the preparation of
specimens of parttally purified virus lysates for quantitative electron mtcrographtc
analysis. YzroIogy l&359-371.
2. Mittereder, N., March, K. L., and Trapnell, B. C (1996) Evaluatton of the concentration and bioactivity of adenovirus vectors for gene therapy. J Vlrol. 70,
7498-7509.
3. Green, M. and Pina, M. (1963) Biochemical studies on adenovnus multiplication. IV.
Isolation, purification, and chemical analysis of adenovuus. Virology 20, 199-207.
4. Green, M. and Wold, W. S. M. (1979) Human adenoviruses: growth, purificatton,
and transfection assay. Methods Enzymol. 58,425-435.
5. Hashimoto, S., Sakakibara, N., Kumai, H., Nakai, M., Sakuma, S., Chiba, S., and
Fujinaga, K. (1991) Fastidious human adenovirus type 40 can propagate efficiently and produce plaques on a human cell line, A549, derived from lung carcinoma. J Vwol. 65,2429-2435.
6. Green, M., Pma, M., and Kimes, R. C. (1967) Biochemical studies on adenovirus
multiplication.
XII. Plaquing efficiencies of purified human adenoviruses. Discussion and preliminary reports. Vlrohgy 31,562-565.
7. Tollefson, A. E., Scaria, A., Hermiston, T. W., Ryerse, J. S., Wold, L. J., and
Wold, W. S. M. (1996) The adenovirus death protein (E3-11.6K) is required at
very late stages of infection for efficient cell lysis and release of adenovirus from
infected cells. J. Viral 70,2296-2308.
8 Tollefson, A. E., Ryerse, J. S., Scaria, A., Hermiston, T. W., and Wold, W. S. M.
(1996) The E3-11.6kDa adenovirus death protein (ADP) is required for efficient cell death: characterization of cells Infected with adp mutants. Virology
220, 152-162.
Ann E. Tollefson,
1. Introduction
The E3 transcription umt of the well-studied subgroup C adenovtruses (Ad)
(prototypic serotypes 2 [Ad21 and 5 [Ad5]) is located between map units 7686 (see Fig. 1). The E3 region 1ssurrounded by the genes for virion protein
VIII and fiber, and is transcribed off the r-strand. The E3-region transcription
unit is complex Extensive sphcmg controls expression of seven identified E3
proteins, four of which have functions that modify the host immune response
to the viral mfection (reviewed m ref. I, Table 1)
Human adenovirus has served as a model system for studying persistent
infections and has enjoyed widespread interest as a gene therapy vector. In
both cases,understanding the immune response to the virus is paramount for
further advancements m these fields. This will require mutational analysis of
the immune-response modifying E3 region and its proteins, their reincorporation mto the viral genome, and study of the viral infection in vwo A series of
protocols will be presented that have been used for the creation of adenoviruses
with mutations in the E3 region A method detailing the tsolation of adenovirus
DNA containing the terminal protein (TP-DNA) will be described (8-10). This
will serve as the startmg material into which the mutated E3 region will be
inserted, either by overlap recombmation (11) or by ligation insertion (12). In
either method, transfection of the DNA into the cell is required for infectious
virus to be generated, and a protocol will be given. Lastly, a procedure will be
provided that can be used to screen for the desired E3-mutated adenovnus.
Mutagenesis techniques will not be discussed here but excellent reviews are
available (13,14).
From
Methods
m Molecular
Medune,
Vol
21
11
Adenovws
Methods
and
NJ
Protocols
12
Elb
E3 )
EC0
RI
76
20
I
40
I
60
I
l?coRl
83
I
so
I
IE4
4 E2a
ECORI100
a
Ad5 TP-DNA
r%
Mutoled
Ad2
Mulated
Ad2
83
Em RI m.w,c,ion en? me
digeuion 0r ~d5 w- L NA
Ad5
EcoRl
76 Ad2
oR1
Ad5. Ad 2- Ad5
E3 mutated adenovnn
83 Ad 5
D I
IlkoR
B
v
plaques
Fig 1 Methods used to insert a mutated E3 region mto the viral genome (A) Schematic tllustratmg some of the adenovirus transcription units expressed during early
stages of infection (B) Construction of E3 mutations Ligation method. Cleave cloned
Ad2 EcoRI-D fragment contammg a mutation with EcoRI, then ligate to EcoRIcleaved Ad5 TP-DNA complex. Transfect legation mixture mto A549 cells and allow
plaques to form. Overlap recombmatton method. Cotransfect cloned Ad2 KpnI-A fragment containing a mutation m the E3 region with EcoRI-cleaved Ad5 TP-DNA complex into A549 cells and plaques were allowed to form
2. Materials
1 CsCl-purified adenovtrus from 6 L of KB cells (preparation described m
Chapter 1)
2. 8 M GuHCI: aqueous solution (Stgma, St. LOUIS, MO, cat. no. G9284). 8 M
GuHCl (guamdme HCl) can be made up from a powder (Sigma cat no G4505)
m ddH20/2 WPefabloc
(Boehrmger Mannhetm, Indtanapolls, IN) Microwave
400 mL ddH,O to near botlmg, add 764 2 g GuHCl, and stir Adjust volume to 1 L
by the addition of Pefabloc to 2 mM and ddH20. Use this stock for lys~s of adenovuus virions and to generate 4 M GuHCl.
3. Pefabloc: Boehringer Mannheim cat no 1 429 876
Table 1
Location
Sequence
Coding sequence
Ad5 (2)
E3 protein
ACQ (3)
start
stop
start
stop
12.5 K
67K
gp19 K
27,858
28,547
28,735
28,179
28,736
29,215
27,919
28,630
28,812
28,220
28,8 19
29,289
11.6 K (ADP)
29,49 1
29,770
29,468
29,77 1
29,784
30,057
29,78 1
30,054
14.5 K (PYIDP)~
14.7 K
30,062
30,453
30,458
30,837
30,059
30,444
30,449
30,828
Function
Unknown
Unknown
Inhibits Ad-specific cytotoxic T-lymphocyte recognition
and killing of the virally infected cell by binding
MHC class I molecules and inhibtting their transport
to the cell surface
Required for efficient cell lysis and release of adenovuus
from Infected cells ($5)
10.4 K and 14.5 K proteins form a complex that.
Prevents TNF cytolysts
Clears epidermal growth factor and msulm receptors
from the infected cell surface
Clears Fas antigen from the mfected cell surface (6)
Inhibtts TNF-mduced apoptosis
GTPase bmdmg protein (7)
14
10
11
12
13.
14.
15
16.
17.
18.
Adenowrus E3 Mutations
15
19 1 8% Noble agar. 1.8 g ofNoble agar (DIFCO, Detroit, MI, cat no. 0142) m 100 mL
ddH,O Sterilize by autoclavmg
20 Hirt lys~s buffer. 0 6% SDS, 0 01 M EDTA, 0.01 M Tris-HCl, pH 7.4
21. Proteinase K: (Boehringer Mannhelm cat no. 1 373 196) 15 mg/mL stock
22 5 MNaCl.
23 Loading dye. 6X buffer 0 25% bromophenol blue, 0.25% xylene cyan01 FF, 30%
(w/v) glycerol in water, store at 4OC.
24. 0 8% agarose. Electrophoresis-grade agarose (Glbco-BRL, Galthersburg, MD,
ultrapure agarose, cat no 15510-027). Prior to pouring the gel, add ethldium
bromide (Sigma cat. no E875 1) to a concentration of 0.5 clg/mL for DNA visuallzatlon under UV light
25 50X TAE buffer (per liter) 242 g Tris base, 57.1 mL glacial acetlc acid, 100 mL
0.5 M EDTA (pH 8.0), brought up to 1 L by the addition of ddH,O (workmg
concentration of 40 mM Tris-acetate, 2 ti EDTA)
26. Plasmld-containing mutated E3 gene(s) of choice
3. Methods
3.1. Adenovirus TP-DNA Preparation
Adenovlruses contam two origins of replication located in the inverted termmal repeats. The E2-coded terminal protein, along with the adenovirus DNA
polymerase, bind wlthin the origin of replication where the terminal protein 1s
cleaved to a polypeptlde of 55 kDa. This smaller polypepttde is then covalently
attached to the vu-al genome, where it serves to initiate viral DNA replrcatlon.
Purified adenovlrus DNA that has retained the terminal protem produces vu-al
plaques at a 40-100 times higher frequency than viral DNA lacking the
termmal protein followmg transfection (8,9). Because of its enhanced
Infectivity, TP-DNA has been used as a starting point toward constructmg
recombinant adenoviruses mutated in the E3 region. The following IS a protocol for the purification of TP-DNA from the point where the mvestlgator has
CsCl-purified adenovirus. Preparation of CsCl-purified adenovlrus is described
m Chapter 1.
3.7.1. Column Preparation
1 Fill the column with 200 mL of 4 M GuHCl-TEMP.
2. Mix 250 mL of Sepharose 4B with 750 mL 4 MGuHCl-TEMP,
degas for 10 mm.
3 Add the Sepharose 4B/GuHCl-TEMP
solution to the column A glass rod may be
used to stir the Sepharose 4B solution m the column to remove any air bubbles
4 Move the column to 4C and connect the column to a fraction collector Estabhsh
a flow rate of 18 mL/h Elute excess column buffer, being careful to retam enough
column buffer so that the packing material 1s not exposed to air. Allow the
Sepharose 4B to settle (usually overnight) at 4C. The final column bed volume
will be approx 35 cm.
76
3.2. Transfecfion
of A549 Cells
Thts se&on presents a standard protocol used for transfecting A549 cells to
generate recombmants m the E3 region. Mutations m the E3 12.5 K, 6.7 K,
gp19 K, ADP (11.6 K), and the majority of the 10.4 K-coding sequence can be
transferred to the viral genome by hgation of the mutated Ad2 EcoRI-D fragment to EcoRI-digested
Ad5 TP-DNA (12). In cases in which mutations are
needed in the 14.5 K and 14.7 K protems, homologous recombmation has been
Adenovirus
17
E3 Mutations
0.8
0.7
x
0.6 E
X
0
Xo
.o
0
X0
X0
xxxxxxxx
15
0
Y XX
l
x
by;:
A
X
30
X0
xxx
loo
45
* Only an npproxlmatton,
elution times and peok levels WIII
varydependent on column packmg and startmg viral matcrud
Fig 2. Schematic representation of a typical graph of 260 and 280 absorbance readmgs
from an adenovirus TP-DNA isolation using Sepharose 4B column chromatography.
with EcoRI
I). These cells should be plated at approx 5 x IO5 cells per 60 mm dish (2
dishes per viral construction) the day before the transfection. This will put the
cells at 60-80% confluency at the time of transfection. Correspondmg adjustments to accommodate 35- or loo-mm dishes can be calculated accordmgly.
3.2.2. Preparation of DNA
3.2.2.1. LIGATION PROTOCOL (SEE NOTE 2)
1. Calculate the amount of plasmid containing the E3 region mutated gene(s) needed
to generate 10 pg of insert DNA by EcoRI cleavage. Followmg cleavage of the
plasmid, add calf-intestme phosphatase to the reaction, treating for 30 mm at
37C This treatment Inhibits the formation of multtmers m the ensumg step of
ligating the mutated Ad2 EcoRI-D fragment into the EcoRI-cleaved TP-DNA
Gel purify the mutated viral DNA fragment and ethanol precipitate the Insert
DNA to concentrate the fragment.
18
2 Cleave 2.5 ug of Ad5 TP-DNA wrth 20 U of EcoRI per viral construct The
EcoRI cleavage of the Ad5 TP-DNA will create three fragments, 2733 1, 5886,
and 2718 bp m size. Check for adequate cleavage of the viral TP-DNA by running 0 5 pg of the DNA on a 0.8% agarose gel The larger two fragments are
from the termmt of the vnus Because of then association with TP, they ~111 not
migrate mto the gel and will appear retained m the well of the gel followmg
exposure to UV light Smce the EcoRI fragment of 2718 bp IS an Internal fragment, it will migrate easily into the gel The EcoRI-digested Ad5 TP-DNA will
be used directly m the ligation reaction with the mutant viral DNA (Overnight
restriction-enzyme dtgestton usually results m Me detectable restriction endonuclease activtty the followmg day ) Do not ethanol-precipitate the restrtctron
endonuclease-cleaved TP-DNA
3 Combme 2 pg of the EcoRI-cleaved Ad5 TP-DNA with 5 pg of the mutated E3
insert DNA (Ad2 EcoRI-D fragment) and hgate overmght at 16C This puts the
insert at a 33-fold molar excess over the equivalent wild-type sequence that ~111
still be present in the EcoRI-cleaved TP-DNA. The large molar excess, however,
favors the msertion and wild-type vnus IS rarely regenerated (~10%)
3 2 2 2. OVERLAP RECOMBINATION PROTOCOL
In this method, the mutated viral DNA segment 1s not gel purified. Instead,
a plasmtd with the KpnI-A fragment of Ad2 (bp 25,881 to 33,594) containing
the mutant E3 gene(s) 1s cotransfected with the EcoRI-digested
Ad5 TP-DNA
The EcoRI digestion ensures that the wild-type vu-us 1s not regenerated and
retains enough overlappmg sequence for homologous recombmatton
to occur
between the EcoRI-cleaved
Ad5 TP-DNA
and the KpnI-A
adenovtrus
sequences in the plasmtd. This protocol IS similar to the hgatton technique
described previously
but elrmmates the need to isolate the mutated viral
DNA sequence from a plasmtd and the ligatton step, making tt more time
effective. Simply calculate the amount of the E3-mutated plasmid required
to generate 10 pg of the mutated viral DNA segment and cotransfect tt with
the restriction-enzyme-drgested
viral genomtc TP-DNA followmg the protocol described below.
Adenovirus
E3 Mutatrons
79
3 Combme the ligand DNA with 20 clg of salmon sperm DNA and add sterile
ddH,O to a volume of 450 pL
4. Add 50 & of 2.5 M CaCI,.
5 Place 500 pL of 2X HeBS mto a sterile IS-mL comcal tube. While mtxmg the 2X
HeBS (either mechanically by bubbling air through the solution usmg a mechamcal pipettor or by hand shakmg the solution) add the DNAKaCl, solution dropwise
(using a Pasteur pipet or Ptpetteman). Immediately vortex the solutton for 5 s
6 Allow the tube to stand at room temperature undisturbed for 20 mm while a precipitate forms
7 Aspirate the medium off the cell monolayer and add 0.5 mL transfection mixture
onto cell monolayer in each 60-mm dish Incubate for 20 mm m a 37C 5% CO2
mcubator
8 Add 4 mL complete medra (DME + 10% FCS) and Incubate for an additional 4 h
m a 37C COz incubator
20
3.4. hitid
of Viral Plaques
1. Plate A549 cells at 5 x lo5 cells per 35-mm dish the day before picking plaques
This will place the cells at 80% confluency the day of the infection
2. Remove medium from cells and add 0.2 mL vuus (agar-plug suspenston solutton
described in the previous step). Adsorb at room temperature for 30 mm and then
add 2 5 mL complete medta and incubate at 37C. Cells should not need to be
refed during the imtial propagatton.
3 Viruses are ready to harvest when all cells are rounded (due to the viral cytopathic effect or CPE) and most have detached from the dish (usually 4-7 d)
4. To permit collectton of medmm (as stock) while retammg the majority of the infected
cells (for analysis), leave dishes undisturbed in the tissue-culture hood for 30 mm
5 Gently remove 2 mL medium and add tt to a sterile veal containmg 0 25 mL
sterile glycerol Gently mix and store these candidate viruses at -70C
6 Slowly aspirate any remammg medium from the plate If this IS done carefully,
the majority of cells ~111remam m the dish and can be used m the next section for
analysts of adenovirus plaques.
Adenovirus
21
E3 Mutations
A
map units
20
10
30
40
50
60
70
80
76
57
Kpn
23.7
I
39.8 42.8
IIIHI
47.5
31.4
173
I
I
D
E
B
c
61.9
IEI
IGIEIKIJI
7.8
Hand 111
32.2 +.@41.9
1 IlMiJI
I HIJI
D
D
51.0
1
I
72.0
-A
-B
-c
-D
-c
-D
E
Cl9
83.6
89.8
Ad 2
Ad5
Ad 2
Ad 5
-E
-F
-CI
-F
-G
-H
=i
B*
-1
-
762
IFIDIEIC
-A
80.2
Ad5-Ad2-Ad5
El mutant
Ad5
Ad2
Ad5
93 5
IF
71.4
B
100
86
IDIFICIH
59.4
A
Eco RI
90
E3
B*
-J
22
4
5
6.
7
8.
9
10
11
12
13.
4. Notes
1. Transfection reagents: An exact pH for the 2X HeBS solution is extremely
important for efficient transfection There can be wide variability m the efficiency of transfectton obtained between batches of 2X HeBS The efficiency
should be checked with each new batch. The 2X HeBS solution can be rapidly
tested by mixing 0.5 mL 2X HeBS with 0.5 mL 250 ti CaCI, and vortexing
Place a drop onto a glass shde with a cover slip. A fine precipitate should develop
that is readily visible on the microscope. Transfection efficiency must still be
confirmed, but if the solution does not form a precipitate in this test, there is
something wrong Alternatively, kits of these reagents are available commercially from Promega (Madison, WI, cat no. E1200), Sigma (cat. no. CA-PHOS),
and Clontech (Palo Alto, CA; cat. no. K2050-1). The Clontech kit also includes a
reagent, CalPhos Maximizer, which enhances the transfectton efficiency by 3 5to 12.7-fold, dependent on the cell type bemg used. Alternate cell lines may be
used, however, transfectton procedures need to be refined to the cell type used
and an optimization protocol has been described (13)
Adenovirus
E3 Mutattons
23
2 Plasmtd DNA preparatton. The quality of the plasmtd DNA used m transfectton
expertments IS crucral for success. Plasmid DNA should be prepared by CsCl
banding (13) or by commerctally avatlable kits designed to reduce the quantity of
lipopolysacchartde (LPS) present in the completed plasmtd preparation (Qtagen,
Chatsworth, CA, cat no 12362)
3. Plaque assay. The plaque appearance may be greatly delayed (2 1 d or longer) in
cases m which the adenovnus E3 ADP (11.6 K) protein expression is altered by
deletion or mutation These plaques will typically be small in size.
4 Screenmg adenovirus plaques* An alternattve method to determme the E3-mutant
adenovnus utrhzes m vtvo labelmg of the vtral DNA wtth 32P. Much of this procedure parallels that previously discussed with the followmg exceptrons Seven
to nme hours postmfectton, wash the cell monolayer with phosphate-free DMEM
(Gtbco-BRL, cat no 1197 I-025); then add 1 mL of phosphate-free DMEM contaming 2% FBS and 32P-orthophosphate (NEN-DuPont, Wilmington, DE; cat.
no. NEX-053) at 50 @I/ 35-mm plate. Incubate overnight in a 37C CO, mcubator (32P orthophosphate IS a p emitter. The mvesttgators radtatton safety officer
should be nottfied for proper handling and dtsposal of all matertals ) Harvestmg,
processmg, and restrtctton-enzyme digestion of the vtral DNA can contmue as
prevtously descrtbed. After the restrictton-enzyme fragments are separated by
gel electrophorests, the gel can be drted at 80C for 2 h under a vacuum (be sure
to have a cold trap installed to capture 32P-radtoacttve waste) and then exposed to
autoradiography film for 5 mm or more at room temperature
References
1. Wold, W. S. M , Tollefson, A E , and Hermiston, T W (1995) E3 transcriptton
unit of adenovu-us, in The Molecular Repertowe of Adenovwuses, Current TOPICS
zn Mzcroblology and Immunology, vol. 199 (Doerfler, W. and Bohm, P , eds ),
Springer, pp 237-274
2. Chroboczek, J., Bteber, F , and Jacrot, B. (1992) The sequence of the genome of
adenovnus type 5 and tts compartson wtth the genome of adenovnus type 2
Vwology 186, 28&285.
3 Roberts, R. J , ONetll, K E., and Yen. C. T. (1984) DNA sequences from the
adenovnus 2 genome. J Blol Chem 259, 13,968-13,975.
4. Tollefson, A E , Ryerse, J S., Scaria, A., Hermiston, T. W., and Wold, W S. M.
(1996) The E3 11 6-kDa adenovtrus death protein (ADP) is requtred for efficient cell death characterization of cells infected with adp mutants. Vzrology
220,152-162
726-730
24
7. Li, Y., Kang, J., and Horwitz, M J. (1997) Interaction of an adenovirus 14 7kilodalton protein inhibitor of tumor necrosis factor alpha cytolysls with a new
member ofthe GTPase superfamily of signal transducers. J Vzrol 71, 1576-l 582
8 Robmson, A. J., Younghusband, H. B , and Bellett, A. J D (1973) A cucular
DNA-protein complex from adenovnuses. Vzrology 56, 54-69
9 Sharp, P. A., Moore, C , and Haverty, J. L (1976) The mfectrvrty of adenovuus 5
DNA-protein complex VzroZogv 75,442-456
10. Chmnadural, G , Chinnadurai, S., and Green, M. (1978) Enhanced mfecttvtty of
adenovlrus type 2 DNA and a DNA-protein complex J Vzrol 26, 195-199
11 Ranhelm, T. S., Shtsler, J., Horton, T. M., Weld, L. S , Gooding, L R., and Wold,
W S. M. (1993) Characterization of mutants wrthm the gene for the adenovuus
E3 14.7-kilodalton protein which prevents cytolysts by tumor necrosts factor J.
Vzrol 67,2159-2167
12 Wold, W S M , Deutscher, S L., Takernon, N , Bhat, B M., and Magle, S C.
(1986) Evidence that AGUAUAUGA and CCAAGA UGA mmate translation m
the same mRNA m regron E3 of adenovu-us. Vzrology 148, 168-180.
13. Ausbel, F. M , Brent, R , Kingston, R. E., Moore, D D , Seidman, J G., Smtth, J.
A., and Struhl, K (1994) Introduction of DNA into mammalian cells, m Current
Protocols zn Molecular Bzology, vol. 1, Wiley, New York, pp. 9.1 l-9 5 5
14 Trower, M K. (1996) A protocol for site-directed mutagenesis employmg a uracrlcontaining phagemld template Methods Mol. BIOI 58,469-476.
15 Deryckere, F and Burger-t, H -G (1997) Rapld method for preparing adenovrrus
DNA Bzotechnzques 22, 868-870
3
Isolation, Growth, and Purification
of Defective Adenovirus Deletion Mutants
Gary Ketner and Julie Boyer
1. Introduction
Adenovirus mutants that lack essential genes must be grown by complementation, the products of the missmg genes supplied by a source other than the vtral
genome. Two methods are available for the growth of defective adenovirus
mutants by complementation. For mutations confined to E 1, E4, or portions of
E2, complementmg cell lines that contain segments of viral DNA and that can
supply the missing viral products can be used to produce pure stocks of mutant
particles (1-9). This approach will probably be extended to other regions of the
viral genome, but may prove difficult to adapt to genes such as the late genes,
whose products are required in large amounts by the virus. Alternatively, defective mutants can be grown as mixed stocks with a second helper virus that can
supply in truns functions required by the mutant (10). Providing that a mutant
contains all of the c&-active elements required for viral growth and is large
enough to be packaged into an adenoviral capsid, there are in principle no restrictions on the DNA sequencesthat can be deleted from a mutant grown by complementation with helper virus. In addition, becausethe helper virus replicates, even
products needed in large amounts can be effectively supplied in trans. Recently,
adenoviral genomes constructed for gene therapy purposes and lacking nearly all
viral sequenceshave been propagated in this way (11-13).
Growth of mutants by complementation with helper virus requires that, for
most purposes, the mutant and helper be physically separatedbefore use. This is
done by CsCl equilibrium density gradient centrifugation. The following protocols were developed for propagation of defective mutants with modest deletions
(l O-20% of the viral genome), but are applicable to larger deletions and to substitutron mutants with genome sizesthat differ from that of wild-type virus.
From
25
and Protocols
NJ
26
1.2. Selection
of Helper Virus
3. The helper should have no properties that make low levels of contammation
the eventual purified mutant stock unacceptable,
scheme IS completely effective.
as no physical
of
purification
2. Materials
1 CsCl solutions, All CsCl solutions should be 20 mM Trrs-HCl (final concentration), adjusted to pH 8.1. Adjustment of the pH should be made after dissolvmg
the CsCl as some lots produce very acidic solutions.
Defective Adenovirus
2.
3.
4.
5.
Deletion Mutants
27
CsCl density 1.25, refractive index 1.3572; 33.8 g CsCl per 100 mL of solution.
CsCl density 1.34, refractive index 1.3663; 46.0 g CsCl per 100 mL of solution.
CsCl density 1.7: refractive index 1.3992; 95.1 g CsCl per 100 mL of solution.
1,1,2 Trichlorotrifluoroethane
(Sigma, St. Louis, MO; cat. no. T5271).
200X IGEPAL (Sigma cat, no. I-3021): 10% IGEPAL CA-630 solution m water.
TE. 10 mA4Tris-HCl, 1 mMEDTA, pH 8.1.
PBS: per liter: 160 g NaCl, 4 g KCI, 18.2 g Na*HPO,, 4 g KH,PO,, 37.2 g EDTA.
pH should be 7.2.
3. Methods
3.7. lsolation of Defective Mutants
by Complementetion
PIaquing
3.7.1. With Defective Helpers (see Notes 1 and 2)
1. Prepare host cell monolayers m 6-cm tissue culture dishes. If applicable, the host
cells should be nonpermissive for the helper,
2. Determine the number of cells in one dish.
3. Transfect monolayers with mutant DNA by the calcmm phosphate procedure
(protocol elsewhere in this volume). DNA fragments can be used if mutants are
being constructed by a recombinational strategy.
4 After transfection, infect the monolayer at an MO1 of 2-5 PFU/cell with the
helper virus. Remove the medium from the transfected dishes, add the virus
m 1 mL medium, adsorb for 2 h, remove the inoculum, and till the dishes
with agar medium.
5 Contmue by a standard plaquing protocol. If the helper is a ts mutant, incubate
the dishes at the restrictive temperature.
6. When plaques are vistble, pick and screen for the presence of the mutant (see
Chapter 4)
Helpers
28
3. Pipet 0.1-0.25 mL of a mixed moculum onto the lower edge of the monolayer.
The inoculum can be a ministock (Chapter 4), a portion of a previously made
mixed stock, or a stock enriched for the deletion mutant by one round of CsCl
denstty gradtent centrifugation (below), diluted with medium to approx log
infectious units per mL.
4. With the dish still tilted, incubate at 37C in a humldlfied incubator for 2 h.
(Proper humidification IS important to prevent the raised side of the monolayer
from drying out.)
5 Remove the moculum, refill the dish with medmm, place the dish flat, and mcubate until all of the cells have detached from the plate. If a ts helper is being
used, incubate at the restrictive temperature. Feed twice weekly by replacement of the medium until evidence of viral infection 1s seen over a substantial
portion of the monolayer.
6. Harvest the infected cells and medium when all of the cells have detached from
the dish. This mixed stock can be stored at -80C until use.
mutations (> 10%) can be efficiently purified from helpers with a wild-type
genome size by this method, although helper virus with longer than wildtype genomes have been used to make the purification of mutants with smaller
deletions possible (14).
1. Prepare a mixed lysate. One dish should be labeled with 32P as described below.
2. To the mlxed stock, add IGEPAL to a final concentration of 0.05%.
3. Extract the stock vigorously with l/5 volume of 1,1,2 trichlorotrifluoroethane. Recover
the aqueous phase after centrifugation at 4000g for 5 min in a Sorvall GSA rotor.
Re-extract the cell debris and organic phase with a small volwne of PBS; recover
the aqueous phase and pool with the supematant from the previous centrifugation
4. Prepare a discontinuous CsCl gradient in a 35-mL polypropylene centrifuge tube
by adding (in order) approx 20 mL extracted virus suspension, 4 mL. CsCl density 1.25, and 5 mL CsCl density 1.7. Add each solution slowly through a plpet
placed all of the way to the bottom of the tube. After the CsCl solutions have
been added, fill the tube to the desired level with extracted vn-us suspension.
5. Centrifuge for 90 min at 17,000 rpm (29,000g) (Sorvall SV288 rotor or equivalent) or 3 h at 25,000 rpm (82,000g) (Beckman SW27 rotor or equivalent).
6. In a darkened tissue culture hood, illuminate the gradient wrth a narrow beam of
light from one side. A microscope lamp is a suitable light source. The virus will
form a sharp, blue-white, translucent band at the interface of the two CsCl solutions. A broader, yellowish or tan, frequently granular layer of cell debris will
appear at the top of the lighter CsCl cushion.
Defective Adenovirus
Deletion Mutants
29
7. Collect the virus, avoiding the cell debris (see Note 3).
8. Adjust the concentrated virus suspension to a density of 1.34 (refractive index
1.3663) with 20 mM Tris-HCl, pH 7.5 or with the density 1.7 CsCl solution.
Adjust to the required volume with CsCl density 1.34.
9. Centrifuge the suspension at 35,000 rpm for 16 h in a Sorvall TV865 rotor (or
equivalent). Two closely spaced virus bands should be visible m the center of
the tube.
10. Fractionate the gradient into single-drop fractions through a hole made in the
bottom of the tube.
11. Measure the radioacttvity m each fraction by Cherenkov counting. Two more or
less well-separated peaks should appear (Fig. 1).
12. Pool the fractrons that comprise the lighter peak (see Fig. 1) and repeat steps
8-11 (see Note 4).
13 Estimate the infectious titer of the virus suspension from its A,,, An A,,, of 1
corresponds to a plaque-formmg titer of 3.5 x 1OgPFU/mL for Ad5 purified over
three CsCl gradients.
14. The purity of the mutant stock can be assessed by plaqumg under conditions
permissive for the helper, or by restriction enzyme digestion of purified DNA.
15 Purified virus is stable for months m buoyant CsCl at 4C. However, htgh-titer
suspensions dialyzed against solutions of low ionic strength (for example, TE)
frequently precipitate. If it is necessary to remove the CsCl from a purified stock,
first adjust the A 260of the suspension to 0.5 or lower, and mimmize storage time
at low ionic strength.
3.4. Preparation
of 32P-Labeled
4. Notes
1. Some defective vrruses kill cells that they infect even though they do not form
plaques. If monolayers infected with helper at the MO1 recommended here do
not survive, the following modification should be used
a. Prepare monolayers m 24-well tissue culture dishes.
30
40
40
Fraction Number
2.
3.
4.
5.
31
0.5 mL of medium, adsorb for 2 h, remove the inoculum, and refill with
medium. If the helper is a ts mutant, incubate at the restrictive temperature (see Note 2)
e. 12-16 h after infection, trypsinize each well and reseed the transfected/
infected cells in a 6-cm dish along with enough uninfected cells to form a
confluent or nearly confluent monolayer.
f. After the cells have attached (8-24 h), overlay with agar medium and proceed
as described In Subheading 3.1.1., step 5.
If the mutant is available as virus particles (as in the isolatton of naturally occurrmg mutants), use one of the protocols in Subheading 3.1.1., or m Note 1,
replacing the transfection step with infection by the mutant stock at approx 50
mfectious particles per dish (or well).
It is convenient to collect virus through a hole made m the bottom of the tube
with a pushpin. Plug the tube with a rubber stopper pierced by a large-gage
syringe needle, close the needle with a finger over its hub, and make the hole m
the bottom of the tube. The rate at which liquid flows out of the hole can be
controlled by finger pressure on the hub of the syringe needle. Alternatively, a
needle can be inserted through the side of the tube and the virus band drawn out
with a syringe. For lysates from two or more lo-cm dishes, the virus band should
be visible in the drops as they leave the tube; for small preps, 32P-labeled virus
tracer (see above) can be added before centrifbgation and fractions can be collected and the virus located by Cherenkov counting.
Depending on the purity required, two or more gradient steps may be necessary.
In the experiment shown in Fig. 1, contamination of the deletion mutant stock by
helper was approx 0.03% after three gradient steps.
If labeled virus to be used as tracer 1s being prepared in parallel with a large
unlabeled stock, the labeled cells should be harvested at the same time as the
large stock, even if not all cells appear to be infected. If the labeled dishes are ready
for harvesting before the remaining dishes, collect and rinse the cells and
store at -80C until needed
References
1. Graham, F. L., Smiley, J., Russel, W. C., and Nairn, R. (1977) Characteristics of
a human cell line transformed by DNA from human adenovirus 5. J. Gen. Vzrol.
36, 59-72.
2. Weinberg, D. H and Ketner, G. (1983) A cell line that supports the growth of a
defective early region 4 deletion mutant of human adenovirus type 2. Proc Natl.
Acad Sci. USA 80,5383-5386.
3. Brough, D E., Lizonova, A , Hsu, C., Kulesa, V. A., and Kovesdi, I (1996) A
gene transfer vector-cell line system for complete functional complementation of
adenovirus early regions El and E4. J. Viral. 70, 6497-6501.
4. Brough, D. E., Cleghon, V., and Klessig, D. F. (1992) Construction, characterization, and utilization of cell lines which inducibly express the adenovints DNAbinding protein. Virology 90,624-634.
32
8. Wang, Q., Jia, X.-C,, and Fmer, M. H. (1995) A packaging cell lme for propagation of recombinant adenovirus vectors containing two lethal gene region delettons. Gene Ther. 2,775-783.
9. Yeh, P., Didieu, J.-F., Orsim, C., Vigne, E., Denefle, P., and Perricaudet, M
(1996) Efficient dual transcomplementation
of adenovirus E 1 and E4 regions
from a 293-derived cell line expressing a minimal E4 functional unit. J Vzrol
70,559-565.
13. Kochanek, S., Clemens, P. R., Mitani, K , Chen, H -H., Chan, S., and Caskey,
C. T. (1996) A new adenoviral vector: Replacement of all viral coding
sequences with 28kb of DNA independently
expressing both full-length
dystrophin and P-galactosidase Proc. Natl. Acad. Scz USA 93, 573 l-5736.
14. Falgout, B. and Ketner, G. (1987) Adenovirus early region 4 is required for efficient virus particle assembly. J. Virol 61, 3759-3768
33
and Protocols
NJ
34
Fig. 1 (A) The orgamzatlon of adenovuus early regton 4 E4 open reading frames
(ORFs) are indicated by open boxes above a scale that Indicates position m map units
(0 to 100; 1 map unit is approx 360 bp) and nucleotide numbers. ORFs 1,2, 3,4, and
6 are colmear with the viral DNA; ORFs 3/4 and 6/7 are the result of inframe sphcmg
of the E4 mRNA precursor E4 is transcribed from right to left, (B) Reconstructton of
an intact viral genome by ligation in vitro (C) Reconstructton of an intact viral
genome by overlap recombmation. (D) Construction of an E4 mutant by recombmation between a plasmid and an E4 deletion mutant In C and D, the X indicates a
potential homologous recombmation event. In B, C, and D, the position of a hypothetical E4 mutation is noted by a V
use of complementtng
to recover.
35
contammg 5 mtrons. Then modes of actton apparently differ; ORF3 specifically stimulates the accumulation of messagescontaining optional exons such
as the adenovnus 1 leader, whereas ORF6 stimulates accumulatton of messageswith or without such exons. The Elb 55-kDa protein is not required, nor
are viral nuclerc acid sequences required m the target RNAs (10,11,39). The
ORF6 product has roles m the transport of late viral mRNAs from the nucleus
to the cytoplasm of infected cells (81 and 1srequired for completion of secondstrand DNA synthesis m the life cycle of AAV (12). ORF6 forms a physical
complex with the 55-kDa product of adenovnus early region 1b (E 1b) and the
complex is the functtonal unit m at least some steps in late gene expression
(13-15). The ORF6 product also bmds the cellular p53 protein, preventing
activation of transcription by p53 in transfection systems (26). Whether this
activity is mvolved in late message production is not known. Both ORF3 and
ORF6 affect the architecture of the nucleus in infected cells and may mediate
some of then effects in that way (I 7,181.
ORF4 mutants are viable m standard hosts. Expression of ORF4 depresses
transcription from at least some promoters that contam CREB protem-bmdmg
sites, mcluding cJunB (19) and the adenovirus E2 and E4 promoters (20,21)
The effect of ORF4 is indirect; an ORF4-induced change in the phosphorylanon state of cFos results m a reduction m cJunB transcriptton and consequently
JunB levels and AP- 1 activity. Reduced AP- 1 activity presumably reduces E2
transcription (21) The ORF4 protein bmds to and modulates the activity of
protein phosphatase 2a, providmg a plausible btochemical mechamsm for its
effect on cFos phosphorylation (22). As a result of its effects on E2 transcription and, perhaps, on protein phosphorylatton, ORF4 downregulates viral DNA
synthesis. This activity IS antagomzed by the E4 ORF 3 and 6 products by an
unknown mechanism (23).
ORF6/7 mutants are viable m standard hosts. The ORF6/7 product binds to
transcription factor E2F and confers cooperattvity on E2F binding to promoters, such as the E2 promoter, that contain appropriately positioned E2F bmding sites (24,225). As a consequence, ORF6/7 expression stimulates E2
transcription in infected cells (26). In most host-cell types, this effect requires
the E 1a 289R protein, which dissociates E2F from pRB and makes it available
for binding by ORF6/7 (27). ORF6/7 mutants are not obviously defective m
viral DNA synthesis in cultured cells.
1.1. Complementing
Cell Link
E4 mutants lacking both ORF3 and ORF6 are defective for growth m normal adenovirus hosts and must be propagated on cell lines capable of supplying required E4 functions in trans. The most commonly used such cell lme is
W 162 (9). The W 162 cell lme was constructed by mtroducmg the Ad5 EcoRI
36
B fragment (map units 86.3-100) mto Vero cells as part of a plasmrd carrying
the selectable marker gpt. W 162 cells contain intact E4 and supply the E4 functions required to support the growth of deletion mutants lacking all identified
E4 products. E4 is attached to its natural promoter in W 162 cells and it is
presumed that E4 expression 1soff until induced by Ela expression from an
infecting virus. Both WI62 and Vero cells (which are of monkey origin but
permissive for human adenovnuses) support plaque formation by wild-type
adenovirus approx lo-fold less efficiently than do other adenovnus hosts, such
as 293 cells. It IS therefore important when comparing defective E4 mutants
titrated on W 162 cells to other viruses on a PFU basis, that W162 titers be
obtained for all stocks.
In addition to W 162 cells, several 293-derived cell lines that complement
mutants with lesions m both El and E4 have been isolated. These lines, developed primarily for use in construction of gene-delivery vectors, make it possible to grow multiply-defective mutants Because of its role m mhtbtting
host-cell protein synthesis, E4 IS presumed to be toxic if constitutively
expressed. Ela expression induces the E4 promoter, and regulable heterologous promoters therefore have been used to control E4 expression in each of
the 293-derived complementing cell lines. E4 expression thus remains low until
it is deliberately induced. The E4 complementing cell lines described so far are
listed in Table 1.
1.2. Isolation of Mutants
7.2.1. Naturally Occurring Mutants
The first E4 mutants were isolated from a stock of adenovnus 2 that had
accumulated a substantial proportion of deletion mutants, presumably as a
result of many undiluted serial passages. All of the mutants lacking E4
sequences were defective and were originally cloned and propagated by the
use of a LShelper virus. For characterization, deletion-mutant parttcles were
purified from the resulting mixed stocks by repeated CsCl density gradient
centrifugatton (ref. 29; see Chapter 3). The development of E4 complementmg
cell lines made the use of helper viruses unnecessary, and most of what is
known of the phenotypes of E4 mutants has been learned using mutants constructed in vitro and propagated on W 162 cells.
1.2.2. Construction of Mutants in Vitro
Most E4 mutants are made by in vitro manipulation of E4-containing plasmids and subsequent mtroduction of the mutations into an intact viral genome.
The mutations themselves can be made by any of a large variety of standard
technrques. Three methods have been used to incorporate E4 mutations into
37
Cell Lines
Mutations
complemented
W162
E4
Promoter/inducer
(if applicable)
EWnone
El, pIX, E4
MMTVldexamethasone
IGRP2
A2
El, E4
El, E4
MMTVIdexamethasone
Metallothtonein/metal
293-E4
2-3-N3
El, E4
El, E4
Alpha mhibin/cAMP
MMTV/dexamethasone
Cell line
Ref.
Weinberg and
Ketner (9)
Kroughak and
Graham(33)
Yeh et al (34)
Ions
Brough et al (35)
Wang et al (36)
G. Kitchmgman,
unpublished
2 1. LIGATION IN VITRO
A variety of viral and plasmid DNA fragments have been used for constructron
of E4 mutants by ligation (Table 2). Both two-fragment and three-fragment
schemeshave been used. In most cases,a large restriction fragment covering the
left-hand portion of the genome, derived from virion DNA or from virion-dertved
DNA-protein complex (DNAPC; ref. 30), 1sligated to smaller plasmid-derived
fragments that make up the remainder of the genome. The ligated DNA is then
introduced into appropnate cells by transfection. The virion DNA fragment can be
purified; alternatrvely an unpurified fragment can be used if the chanceof reassembling parental virus 1sreduced by digesting the vnion DNA with an enzyme that
cuts the right-hand end of the genome repeatedly. Neither of these approaches 1s
completely effective m eliminating plaques produced by virus with the genotype of
the left-end donor and plaques that contain the desired recombinant are usually
mixed. Therefore, plaques must be screenedto identify thosethat contam recombinants and recombinants usually must beplaque purified. Constructrons expected to
yield defective E4 mutants must be done m complementmg cell lines
1.2.2.2.
OVERLAP RECOMBINATION
38
Table 2
Construction of E4 Mutants by Ligation and Overlap Recombination
Virton DNA fragment
Cloned fragment(s)
Ligation
CM0 0 (BarnHI)
60.0 (BarnHI) to 83 6
fromAd DNA PC
(EcoRI) from pBN27,
83 6 (EcoRI) to 100
from pEcoRIBAd5
O-76.0 (EcoRI)
76.0 (EcoRI) to 100
of H5wt300
from ~75-100
O-76.0 (EcoRI)
None
of HW343,76
0
(EcoRI) to 100
from H5dl356
Overlap recombination
t&93 5 (x&21)
76 0 (EcoRI) to 100
of HSd13 1OXba+
from ~75-100
O-79.6 (XbaI)
60 0 (BarnHI) to 100,
from H5wt300
79 6 @&I) to 84.8 (XbaI)
deleted m constructton
None
0 to 76.0 (EcoRI), 73 2
(findII1) to 89 0 (kzzndIII),
83 6 (EcoRI) to 100
Ref
Brtdge and Ketner (6)
Hemstrom et al (38)
into cells along with a plasmrd bearing a mutant version of E4. The mutant
plasmrd must share sequences with the left-hand genomrc fragment(s) so that
homologous recombmation m the region of overlap can regenerate a full-length
viral genome. Consrderatrons such as purrficatron of the left-hand fragment,
the necessity for screening plaques, and the use of complementmg cell lines
are the same as those described for the hgatron protocol, above.
1.2.2.3.
RECOMBINATION
DNA
Many E4 mutants are viable in normal adenovirus host cell lines. Such
mutants can be can be selected after cotransfectron of normal hosts (such as
293 cells) with a mutant E4 DNA fragment and intact viral DNA prepared
from a E4 deletion mutant that will not grow m those cells (for example,
H5d11011, ref. 6). Because no plaque-forming virus IS involved m this protocol, the background of unwanted plaques is much lower than with the above
methods, and when this technique can be used rt IS probably the most efficient
of the available approaches.
39
2. Materials
1. TE: 10 mM Tns-HCl, 1 mA4 EDTA. Adjust to pH 8.1.
2 Buffered 8 h4 guamdmmm hydrochloride solution: 8 A4 guanidinium hydrochloride, 50 mM Tris-HCl, pH 8.1.
3 Guanidimum hydrochlonde/CsCl solutions: 4 Mguamdmmm hydrochloride contaming either 3.03 M CsCl (51.0 g of CsCl per 100 mL of solution) or 4.54 M
CsCl (76 5 g of CsCl per 100 mL of solution), buffered with 20 mM Tns-HCl.
Final pH should be 8.1. Confirm the pH before use, as some lots of CsCl produce
very acidic solutions unless neutralized.
4. PMSF* 100 mM Phenylmethylsulfonylflourlde
in isopropanol. Store tightly
capped at -20C PMSF is toxic.
5. HBS (HEPES-buffered salme) 8 g/L NaCl, 0.37 g/L KCl, 0.125 g/L Na2HP0, 2H,O,
1 s/L glucose, 5 g/L N-2-hydroxyethylpiperazine-W-2-ethanesulfomc acid (HEPES),
final pH 7.05. The pH of the HBS 1scntlcal for the successof transfectlons.
6 Herring sperm DNA Dissolve herring sperm DNA in TE at approx 5 mg/mL.
Somcate on ice m 30-s bursts with an immersed probe until the viscosity no longer
changes appreciably with each burst (6-10 bursts). Determine the concentration
of the solution spectrophotometrlcally and adjust to 2.5 mg/mL with TE
7 Plasmlds (see also Table 2).
a pBN27 (6): pBR322 containing Ad5 DNA from map unit (mu) 60 (BarnHI) to
mu 86.4 (NdeI) One map unit corresponds to 1% of the viral genome, or
approx 360 bp.
b pEcoRIBAd5 (28). pBR322 containing Ad5 mu 83 6 (EcoRI) to mu 100 (right
genomic end)
8 Vu-uses
a Adenovlrus 5
b. H5d11011 (6). a defective E4 mutant that lacks Ad5 nt 33091 to 35353
c. H5d13 lOXba+ (7) carries a single XbaI site at mu 93.5
9 Sucrose solutions* 5,20, and 60% sucrose solutions prepared m I MNaCI, 1 mM
EDTA, 10 mM Tns-HCl, pH 8.1.
10 200X IGEPAL (Sigma, St LOUIS, MO; cat. no. I-3021). 10% IGEPAL CA-630
solution in water.
11 Phosphate-free medium. MEM formulated without sodium phosphate can be
purchased from media suppliers (for example, Glbco-BRL, Gaithersburg, MD,
cat. no. 21-097-027) For labeling viral DNA, supplement with 2% fetal
bovine serum (FBS) (dialysis is not necessary) and 40 pCi/mL carrier-free
32P orthophosphate
12 Lysls buffer: 0 6% sodium dodecyl sulfate (SDS), 200 pg/mL Protemase K, 10 mM
Tris-HCl, pH 8 1, 1 mM EDTA.
13 5 A4 Ammonium acetate
14. 1,1,2 Trichlorotrifluoroethane
(Sigma cat. no. T527 1)
15. 25% glucose solution. 25% (w/v) glucose m HBS; filter sterilize.
16. Agar medium. MEM without phenol red, supplemented with 2% FBS, penicillin,
and streptomycm, and solidified with 0.9% Difco (E. Molesley, Surrey, UK)
40
3. Methods
DNA-Protein
3.2. Construction
of E4 Mutants by Ligation
41
3.3. Construction
of E4 Mutants by Overlap
Recombination
(7)
1
2
3
4
42
Transfection
(31,32)
3.6. Screening
for Mutants
43
4 24 h after infectton, remove the medmm from the infected cells, rinse the monolayer once wtth medium lacking phosphate, and add 1 mL of phosphate-free
medmm containing 2% FBS and 40 pCi/mL 32P orthophosphate.
5. Incubate 6-24 h at 37C
6 Remove the labeling medium and gently rinse the monolayers once with phosphate-buffered salme
7 Lyse the labeled cells by adding 300 pL 0.6% SDS, 200 ug/mL Protemase K m
TE directly to the wells
8. Seal the plate with Parafilm and incubate at 37C 2 h to overnight
9 Transfer the contents of each well to a microfuge tube, add 200 pL of 5 Mammomum acetate and mtx well.
10 Add 1 mL isopropyl alcohol and mix well.
11 Centrifuge m-mediately for 5 mm at full speed m a mtcrocentrtfuge
12 Rinse the pellet twtce in 70% ethanol.
13 Air-dry the pellet and resuspend the DNA in TE
14 The labeled DNA can be analyzed by restrtctton dtgestton, agarose gel electrophorests, and autoradtography for the presence of the mutation
15 After tdenttficatton of plaques contammg the desired recombinants, use the mmtstock
(step 1) for a second round of plaque punficatton. Before plaqumg, add IGEPAL to
0.05% and extract the stock vtgorously wtth l/l0 volume of 1,1,2 tnchlorotnfluoroethane This dtssoctates clumps of vnus that otherwise make plaque purtficatton
extremely Inefficient. Plaques should be purified one round beyond the point where
parental (Ad5 or H5d11011) DNA can no longer be detected m any plaque
4. Notes
1 DNAPC has a tendency to bind to the walls of tubes, and other labware, and so the
digestion of the DNAPC and hgatton should be carried out in the same tube tf possible
To preserve the terminal protem, do not treat the digest with protease, phenol, or SDS
2. Almost all plaques will contam both recombinant virus and H5d11011, the latter
complemented by the viable recombinant When preparing mmtlysates for screenmg, use cells which are nonpermtsstve for H5dflOll to enrtch the mmistocks for
the recombinant However, labeled DNA should be prepared m W 162 cells,
which permit growth of H5d11011 and thus maximize the sensitivity ofthe screenmg procedure for contaminatmg parental virus.
3 Ttmmg of the glucose boost is important for good efficiency and monolayer survival; If multiple dishes are to be boosted as a group, stagger the addition of
the glucose solutton so that prectse timmg can be mamtamed To stmplify the
mampulatton of large numbers of dashes, dishes can be refilled with culture
medium after the glucose boost and overlaid with agar later as a group
References
1. Fryer, G. A., Katoh, Y , and Roberts, R. J (1984) Charactertzatton of the maJor
mRNAs from adenovirus 2 early region 4 by cDNA clonrng and sequencing
Nucleic Aclds Res 12,3503-3519
44
45
17. Ornelles, D A. and Shenk, T. (1991) Localization of the adenovuus early region
1B 55-kllodalton protein during lytic infection: association with nuclear viral
mclusions requires the early region 4 34-kllodalton protein. J Viral. 65,424-439.
18. Carvalho, T., Seeler, J S., Ohman, K , Jordan, P., Pettersson, U., Akusjarvi, G.,
Carmo-Fonseca, M , and Dejean, A (1995) Targeting of adenovirus El A and E4ORF3 proteins to nuclear matrix-associated PML bodies J. Cell Biol 131,45-56
19. Muller, U., Kleinberger, T., and Shenk, T. (1992) Adenovlrus E4orf4 protein
reduces phosphorylation of c-Fos and E 1a proteins while simultaneously reducing the level of AP-1. J. Vwol. 66,5867-5878.
20. Bondesson, M., Ohman, K , Mannervik, M., Fan, S., and Akusjarvi, G. (1996)
Adenovirus E4 open reading frame 4 protein autoregulates E4 transcription by
mhlbiting ElA transactlvation of the E4 promoter. J Vzrol. 70,3844-385 1
2 1. Medghalchi, S., Padmanabhan, R., and Ketner, G (1997) Early region 4 modulates adenovirus DNA replication by two genetically separable mechamsms
Vwology 236, 8-l 7.
22. Klemberger, T and Shenk, T. (1993) Adenovn-us E4 ORF4 protein bmds to protein phosphatase 2A, and the complex down regulates ElA-enhanced junB transcription. J ho1 67, 7556-7560.
23. Bridge, E., Medghalchi, S , Ubol, S., Leesong, M., and Ketner, G. (1993) Adenovu-us early region 4 and viral DNA synthesis Virology 193,794-801
24 Huang, M. and Hearing, P (1989) The adenovirus early region 4 open reading
frame 6/7 protein regulates the DNA binding activity of cellular transcrlptlon factor E2F through a direct complex. Genes Dev. 3, 1699-l 7 IO
25. Neill, S. D , Hemstrom, C., Virtanen, A., and Nevins, J. R (1990) An adenovirus
E4 gene product trans-activates E2 transcrlptlon and stimulates stable E2F
binding through a direct association with E2F. Proc Natl. Acad Scl USA 87,
2008-2012.
26. Swammathan, S. and Thlmmapaya, B. (1996) Transactlvation of the E2 early promoter by El A and E4 617 m the context of the viral chromosome. J. Mel Blol
258,736-746
27. Raychaudhuri, P., Bagchl, S. D., Neill, S., and Nevins, J. R. (1990) Activation of
the E2F transcription factor in adenovirus-infected cells mvolves the E 1A-dependent stimulation of DNA binding activity and induction of cooperative bmdmg
mediated by an E4 gene product. J Vwol. 64,2702-27 10
28. Berkner, K. L. and Sharp, P. A. (1983) Generation of adenovuus by transfectlon
of plasmids. Nucleic Acids Res. 11,6003-6020.
29. Challberg, S. S. and Ketner, G (1981) Deletion mutants of adenovuus 2. lsolatlon
and initial characterization of virus carrying mutattons near the right end of the
viral genome Vzrology 114, 196-209.
30 Robmson, A J. and Bellett, A. D. J (1974) A circular DNA-protein complex
from adenovnuses and its possible role m DNA replication. Cold Spring Harbor
Symp Quant Blol 39,523-53 1.
3 1. Graham, F. L and van der Eb, A J. (1973) A new technique for the assay of
infectivity of human adenovn-us 5 DNA. VwoEogy 52, 45&467.
46
of
by DMSO
33. Kroughliak,
V. and Graham, F (1995) Development of cell lines capable of
complementmg El, E4, and protem IX defective adenovlrus type 5 mutants
Human Gene Ther 6, 1575-1586
34. Yeh, P , Dldieu, J -F , Orsini, C , Vlgne, E , Denefle, P , and Perricaudet, M
( 1996) Efficient dual transcomplementatlon of adenovlrus E 1 and E4 regions from
a 293-derived cell line expressmg a minimal E4 functlonal unit J Vzrol 70,
559-565.
35. Brough, D E , Llzonova, A , Hsu, C., Kulesa, V. A , and Kovesdl, I (1996) A
gene transfer vector-cell lme system for complete functional complementatlon of
adenovirus early regions E 1 and E4 J. Vwol. 70,6497-650 1.
36 Wang, Q., Jla, X.-C., and Finer, M H. (1995) A packaging cell lme for propagation of recombinant adenovuus vectors containing two lethal gene region deletions Gene Ther 2,775-783
37 Marton, M., Balm, S. B., Ornelles, D. A., and Shenk, T (1990) The adenovnus E4
17 kllodalton protein complexes with the cellular transcription factor E2F, altermg its DNA-binding properties and stlmulatmg E 1A-Independent accumulation
of E2 mRNA. J Vwol 64,2345-2359.
38 Hemstrom, K., Nordqvlst, K., Pettersson, U., and Vutanen, A (1988) Gene product of region E4 of adenovuus type 5 modulates accumulation of certain vu-al
polypeptides. J Vu-01 62,3258-3264.
39. Nordqvlst, K and AkusJarvi, G. (1990) Adenovlrus early region 4 stimulates
mRNA accumulation via 5 mtrons. Proc Nat1 Acad Scr USA 87,9543-9547
5
Adenovirus DNA Packaging
Construction and Analysis of Viral Mutants
Susanne I. Schmid and Patrick Hearing
1, Introduction
The selective packaging of adenovnus DNA into a capsid at late times after
viral mfection raises a number of interesting questions. How is the viral DNA
specifically selected from the pool of viral and cellular DNA for encapsidation?
Is the packaging process coordinated with viral DNA replication? What protein-protein and protein-DNA interactions are mvolved in this process? Does
the virus share a packaging mechanism with one or more of the well-characterized prokaryotic phages? The nonenveloped adenovirus particle contams a protein shell consisting of multiple proteins and a DNA-protein core. The
adenovlrus particle contains minimally 12 distinct virus-encoded proteins (1)
(the structural proteins: hexon, penton, fiber, IIIa, VI, VIII, and IX; the core
proteins: V, VII and ~1;and the nonstructural proteins: proteinase and terminal
protein). The virus capsid is assembled by the association of two different
capsomeres: the hexon capsomere, composed of multiple (9-12) hexon molecules, and the penton capsomere, composed of five penton molecules and three
fiber proteins. The hexon capsomeres assemble to form the 20 sides of the
virion particle, whereas the penton capsomeres comprise the 12 vertices of
the virion. The viral core 1s composed of the linear double-stranded DNA
genome, with covalently linked terminal protein at each 5 terminus, in association with three viral core proteins: V, VII, and u.
The assembly of adenovirus particles proceeds through an ordered series of
assembly events and follows the paradigm of prokaryotic phage assembly (2).
The first recognizable viral assembly intermediate is a light-intermedlate
particle (buoyant density of 1.315 g/cc in a CsCl-equilibrium gradient). These
From
47
48
particles contain the capsid structural components and no or very little viral
DNA and associated core proteins. Additionally, light-intermediate particles
contain several proteins that exit the particle during maturation that may represent the adenoviral equivalent of phage scaffolding proteins. The light-intermediate particles mature into heavy-intermediate particles (1.37 g/cc buoyant
density) with the insertion of viral DNA. The heavy-mtermedtate particles
appear to lack core proteins that enter the particle during the next maturation
step with the formation of young virus particles (1.34 g/cc buoyant density).
As the final step in maturation, the virus encoded and encapsidated proteinase
performs numerous cleavages of multiple viral proteins to generate the mature,
infectious virion. Other minor virus-encoded proteins (IIIa, VI, VIII, and IX)
appear to either enhance the assembly of subviral components and/or stabilize
viral protein-protein interactions, and hence particle integrity, once formed.
C&acting packaging sequences in the adenovirus genome are required to
direct selective encapsidation from the left end of the vu-al DNA. Polarity of
adenoviral DNA packaging was mitially demonstrated in studies on viral
mcomplete particles containing viral DNA molecules of subgenomtc length in
which a striking overrepresentation of left-end sequences was revealed, suggesting that DNA packaging occurs in a polar fashion from left to right (3,4). It
was subsequently shown for adenovirus type 16 (Ad16) and Ad3 that a cisacting packaging domain is located within the left 390 bp. The cis-acting packaging domain in Ad5 is located in the left-end 380 bp (5,6). Deletion of the
AdS-packaging domain resulted in virus nonviability, but wild-type growth
could be restored by the substitution of the left-end 353 bp at the right end of
the genome. The AdSpackaging domain is flexible with respect to its position
as well as orientation within certain boundaries. However, it must be located
within 600 bp of the inverted terminal repeat. Detailed analysis of the Ad5packaging domain revealed that it consists of at least seven functional units
called A repeats because of their AT-rich character (Fig. 1). These elements
are functionally redundant, but follow a hierarchy of importance with elements I, II, V, and VI as the functionally most dominant repeats. The
packaging repeats appear to be binding sites for limiting truns-acting packaging factor(s).
The AdS-packaging domain overlaps with two transcriptional enhancer elements (Fig. 1) (7). Enhancer element I consists of a repeated sequence motif
and specifically stimulates transcription of the adjacent ElA region. Mutations
in element I affecting its function can be efficiently complemented by propagation of the virus in 293 cells, a cell line that constitutively expressesthe viral
E 1A and El B gene products (8). Enhancer element II enhances transcription in
cis from all the early transcription units by an unknown mechanism. Element II
mutations result in a decrease in all early transcription, and since some of the
103
49
--
lg4 Enhl
Enhll
Enhl
380
499
50
Comfectlon Exoenment: pat- mutant x wt
cotnfechion
5 PFUkell
I
Harvest
Isolate
Nuclei
Punfy
HMW
cells
2 days
post mfection
1
Isolate
DNA
Punfy
Vtrlons
Vlrlon
DNA
RestmXlon
digestion
Quantltatlon
by Southern
blot
Fig. 2. Schematic outlme of a packaging assay and the results obtained with a
typical packaging-mutant
virus. Input viruses on the top are represented by their
respective genomes. Wild-type genomes (wt) are indicated by white boxes, packaging-mutant
genomes (pat-) by hatched boxes representing the ITR Equal
amounts of replicated mutant and wild-type genomes are present m the nucleus
(middle, left) after coinfection, whereas fewer mutant genomes are packaged
into virion particles than wild-type genomes (middle, right) Relative levels of
mutant and wild type DNA present m the nucleus or in virions respectively are
visualized in then respective lanes (HMW and VIRION) on the Southern blot on
the bottom. Because of the mutation in the pat- packaging domain as indicated
by an arrow on the top, mutant left-end fragments display faster mob&y than
wild-type fragments.
can be normalized
to the total pool of nuclear replicated DNA of mutant
and wild-type virus. This quantitation
of the data measures the reduction of
viral-DNA
packaging independent
of an element II phenotype. Additionally, the coinfecting wild-type virus serves as an internal control. The final
important advantage to this approach is that it is a direct measure of viralgenome packaging.
Adenovirus
DNA Packaging
2. Materials
2.1. Adenovirus
51
Plaque Assay
1. Agar overlay solution no. 1 (for 100 mL): 50 mL 2X DME, 2.5 mL 1 MHEPES, 7.5,
1 25 rnL 1 MMgCl,, 10 mL calf serum, 36 mL 2.8% Difco (Detroit, MI) Bacto-Agar.
2. Overlay no. 2: the same as no. 1 except + 2% serum
3. Overlay no. 3 is the same as no. 2 but also containing 2.5 mL 0.1% neutral red.
2.2. Adenovirus
Particle Purification
2.3. Preparation
of High-Molecular-
Weight DNA
3. Methods
3.1. Construction
and Purification
of Recombinant
Adenoviruses
Plasmids used for all vtrus constructions are derived from pE1 A-WT, which
contains the left-terminal
1339 bp of the Ad5 genome cloned into pBR322 (10).
Deletion and base substitution mutations in pE1 A-WT are introduced into the
packaging domain followmg standard cloning procedures. All mutations constructed in plasmids are rebuilt mto intact viruses by the method of Stow (11).
of d1309 puri-
1. Digest 30-40 pg dE309 DNA overnight using 2-4 U of both ClaI and XbaI
restriction endonucleases per pg viral DNA in a 500~pL reaction volume. A
unique ClaI site is present in d1309 at nucleotide (nt) 918 (2 5 map units [mu])
and a umque XbaI site is present at nt 1339 (3.8 mu).
52
2. Following digestion, layer the reaction over a lo-mL lmear 5-20% sucrose gradient on top of a 1-mL cushion of 60% sucrose in a SW4 1 polyallomer ultracentrif?tge tube.
3. Spin the samples at 10,OOOgusing an SW41 rotor (Beckman, Fullerton, CA) at
15C for 16 h.
4. Collect 1-mL fractions by bottom puncture of the polyallomer tube
5. Examine 5+L aliquots of each fraction on a 1% agarose gel and pool the peak
fractions of the d1309 3.8-100 map unit fragment (usually the first and second
fractions). The smaller left-end fragments are found m the uppermost sucrosegradient fractions.
6. Dialyze the pooled fractions for 4 h at room temperature into TE (see Subheading 2.) to remove sucrose and NaCl.
7. Add one-tenth volume of 3 MNa-acetate, pH 7.4, followed by two volumes of
100% ethanol.
8. Ethanol precipitate the samples, and suspend the final viral DNA in 250 pL TE
for transfection of 293 cells. Approximately 50% recovery of d1309 right-end
fragment should be anticipated and this can be verified by comparison to known
standards on an agarose gel.
Adenovirus
DNA Packaging
53
2. Harvest cells 48-72 h after infection. Resuspend cell pellet in 7 mL TD; place on ice.
3. Sonicate for 30 s on ice, setting 7. Precipitate insoluble material at 5OOOg,4C for 10 min.
4. Layer supernate onto a CsCl step gradient in an SW41 polyallomer tube (2 mL
1.4 g/cc CsCl step, 2.5 mL 1.25 g/cc CsCl). Spin at lOO,OOOg, SW41 rotor
(Beckman) for 60 min at 15C.
5. Remove virion band from 1.25 g/cc: 1.4 g/cc step interface using a syringe and
needle by side-puncture.
6. Reband virions in 1.35 g/cc CsCl-equilibrium
gradient using an SW60 rotor
(Beckman) at 100,OOOg for 18 h at 15C. Remove virion band by side puncture
and either store as purified virions or harvest viral DNA (see below).
3.1.6. Adenovirus
1. Aspirate medium from 293 cell monolayer (60~mm dish), infect with 0.4 mL
adenovirus dilution in TBS + 2% calf serum, at 37OC for 1 h; rock plate gently
every 15 min.
54
55
(single-step
growth) of pack-
56
3. Quantitate virus titers (yield) in the cell lysatesby plaque assayas described in
Subheading 3.1.6.
4. Express the results as the reduction of infectious virus yield of the mutant virus
relative to the wild-type virus. Because of a degree of variability between different virus plaque assays, perform the experiment at least three to five independent
times and calculate the average value and standard deviation for each value.
3. Use the other half of the cells for the isolation of viral DNA as follows. Resuspend the infected cell pellet in 400 pL of 20 mM Tris-HCl, pH 9.0,0.2% sodium
Na-Acetate,pH 7.4, and then ethanol precipitate the DNA by the addition of 2
volumes of 100% ethanol. Store at -2OC overmght, or -70C for 15 min. Spin
the samples at 15,000g for 30 mm to pellet viral DNA Remove ethanol, dry
pellets briefly, and resuspend the viral DNA in 100 pL TE.
9. Use 1 ug of total nuclear DNA and 1/4th the total amount of viral DNA for
restriction-endonuclease digestion and Southern blot hybridization (see Subheading 3.3.).
57
10. Quantitate the levels of wild-type and mutant nuclear and viral DNAs by densitometry (see Note 9) or using a phosphorimager. Calculate the amount of packaged
mutant DNA relative to wild-type DNA, and normalize the levels of packaged
DNA to the levels of nuclear (replicated) DNA of each mutant and wild-type virus.
11. The experiment should be performed at least three to four independent times and
the average value and standard deviation for each value calculated.
4. Notes
4.1. ffeccmsfrucfion
of /?ecombhent
Adenowiruses
58
5 The high-molecular-weight
DNA pellet obtained from approx 1 x lo6 cells is
difficult to resuspend Add TE as indicated above, mix the DNA by repeated
pipettmg, incubate the samples overnight, and repeat the mtxmg The tip of a
Pl 000 tip may be cut off to atd in mtxmg during the mitral resuspension
6. Viral DNA obtained from 1 x lo6 infected cells is generally not visible on an
agarose gel. Prepare all samples side by side, resuspend in equal volumes of TE,
and use 25% of the sample for Southern hybridization.
7. It is very important to verify that the probe is in excess when performmg Southem hybridization analysis. This can be verified by including twofold dilutions of
a standard DNA sample and quantttation of the autoradiogram
8. Generally, packaged viral DNA of packaging-mutant viruses with a reduction m
growth of more than 50-fold in a smgle infection relative to wild-type virus will
not be detectable m the coinfection/Southem hybrtdtzation experiment.
9. For densttometric scanning of the autoradiogram, do not employ an intensifying
screen or use preflashed film Quantitatton of the data can be performed using the
public-domain
NIH Image program (written by Wayne Rasband at the US
National Institutes of Health and available from the Internet by anonymous ftp
from zippy.nimh.nih.gov
or on floppy disk from NTIS, 5285 Port Royal Rd ,
Springfield, VA 22 161, part number PB93-504868)
References
1, Stewart, P. L. and Burnett, R. M. (1995) Adenovnus structure by x-ray crystallography and electron microscopy. Curr Topics Mlcroblol.
Immunol
199/I, 25-38.
2. DHalluin, J. C. (1995) Virus assembly. Curr. Topics MicrobzoE Immunol. 199/l, 47-66.
3. Hammarskjold, M. L. and Winberg, G. (1980) Encapsidation of adenovirus 16
DNA is directed by a small DNA sequence at the left end of the genome. Cell a20,
787-795.
59
10. Hearing, P. and Shenk, T. (1983) The adenovnus type 5 ElA transcriptional control region contains a duplicated enhancer element. Cell 33,695-703.
11. Stow, N. D. (1981) Clonmg a DNA fragment from the left-hand terminus of the
adenovirus type 2 genome and its use in site-directed mutagenesis. J. Vvol 37,
171-180.
12. Jones, N and Shenk, T. (1979) Isolation of adenovirus type 5 host range deletion
mutants defective for transformation of rat embryo cells. Cell 17, 683-689.
13. Wigler, M., Silverstein, S , Lee, L S., Pellicer, A., Cheng, Y. C., and Axel, R.
(1977) Transfer of purified herpes simplex virus thymidine kinase gene to cultured mouse cells. Cell 11,223-232.
14. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Clonzng A
Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
15 Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA
restriction endonuclease fragments to high specific activity. Anal Baochem
132, 6-13.
6
Methods for Creating and Analyzing
Adenovirus Vectors that Express Proteins
that Act on the Viral Genome
C. S. H. Young, Andrea L. Nicolb,
1. Introduction
Adenovirus genetic research has entered a new phase with the recent
upsurge m interest in using the virus as a vector for gene transfer, gene therapy,
and as a vaccine (reviewed in refs. I-3). Although the idea of using adenovirus
as a vector is not new (4J, recent advances both in understanding the functions
of many of the early genes, and in the ability to manipulate the genome to
create the desired genotype, have made it possible to tailor the desired recombinant to very specific purposes. Less attention has been paid to the possibility
of creating adenovirus vectors that express proteins that have direct effects on
the genome of the vector itself, or on coinfecting adenovirus genomes.
Examples of the types of investigation that could be conducted into such trans
effects include:
1. The consequences for the viral replication cycle of overexpressing transcription
viral promoter is controlled, both quantitatively and temporally, and to contribute to the understanding of transcription initiation in mammalian cells in vivo.
Until now, most analyses of viral promoters have involved the creation of mutations in suspected cu-acting elements, followed by inferences about mechanism
from the resulting phenotypes,as measuredm reporter plasmid transient transfectlons or, less frequently, by replacement in the viral genome itself. The ability
to transmit and express either basal or activating transcription factors would be a
considerabletechnicaladvancetoward understandingtranscription initiation during the viral life cycle. So far, the only published reports of the use of adenovnus
From
61
and Protocols
NJ
62
Young et al.
as vectors for transcription factors relevant to the adenovuus life cycle are those
expressing E2F (5) and ~53 (67). Recently, we have constructed vectors
expressing the wild-type form of human TFIIB, and both wild-type and altered
specificity forms of human TBP, and have begun the analysis of the effects of
such expression on the function of the adenovirus major late promoter and on the
overall replication cycle (Lu and Young, unpublished). In principle, the genetic
analysis also could include the use of dominant-negative versions of the factors,
or antisense versions of the RNAs.
2. The consequences of expressing enzymes or proteins thought to be involved in
DNA replication. Adenovirus DNA replication has been extensively characterized in vitro, and it has been demonstrated that at least three cellular proteins are
mvolved in initiation and/or elongation, one being a type I topoisomerase and the
other two being the transcription factors Octl and CTFl (8,9). The biological
importance of the latter two proteins has been suggested by analysis of czs-actmg
element mutations m infection or transfection, but there has been no independent
proof of the involvement of the topoisomerase. Expression of antisense or dominant-negative versions of their respective genes might give definitive proof of
the proteins importance. The genetic analysis also could include the use of factors with altered specificity, or molecules with chimeric DNA binding sites and
functional domains, as have been tested in vitro (10).
3. The effects of creating lesions in the viral genome, such as DNA double-strand
breaks, and the effects of overexpression of enzymes of DNA repair that may be
used to correct them. Although adenovirus has been used extensively to examine
cellular-repair processes, by examining host-cell reactivation of virions treated
with UV or ionizing radiation (e.g., refs. II and 12), standard genetic analysis of
the virus is of limited value because it relies exclusively on the host cell machinery for repair. Nevertheless, overexpression of wild-type and mutant forms of
cellular proteins known or suspected to be involved in repair, could reveal the
way damaged DNA can be corrected. Adenovnus has been used m this way to
examine functional complementation of xeroderma pigmentosum fibroblasts by
the T4 denV gene (13). A recent development is the construction of vectors
expressing endonucleases that cleave the genome of a coinfecting target virus at
specific points (Nicolas, Munz, and Young, in preparation). The fate of the
double-strand break can be followed under a variety of experimental conditions.
4. The effects on adenovirus recombination of overexpression of enzymes or proteins that may be involved m homologous recombmation. Although the initiation
of homologous recombination between viral genomes may depend on the extensive pool of DNA single strands produced during DNA replication, the processing of recombination mtermedrates almost certainly is accomplished by cellular
proteins (reviewed in ref. 24). Recent evidence suggests that mammalian cells
contain homologs of several of the genes of the Saccharomyces cerevwae
RAD52 epistasis group, and that at least some of them are involved m recombination (15). Overexpression of mutant forms or antisense versions of these genes
could be attempted also.
63
This brief and nonexclusive outline suggests that adenovirus vectors could be
a valuable tool for examming various aspects of viral DNA expression and
metabolism, and in turn could lead to insights into fundamental cellular molecular processes. Ideally, the vectors should be designed so that the expression of the
gene product is under the control of the experimenter at all times. In practice this
has not been easy to achieve, and indeed most vectors have been designed to give
continuous and high levels of expression, starting at early times postmfection.
The use of the lox-cre system and other site-specific recombination systems may
allow a much more controlled expression to be achieved (16,17). The designs of
the various expression cassettes described to date have varied in detail, but have
usually involved a strong constitutive promoter and enhancer, with appropriate
splicing and polyadenylation signals. The cassette has usually been embedded in
the experimentally dispensable E 1 or E3 regions of the virus.
In this chapter, the design and methods of construction and analysis of adenovirus vectors expressing proteins that could affect the expresston or the integrity of the viral DNA will be described. Two different vector designs will be
detailed as they give different levels of constitutive expression, but it must be
emphasized that more controlled expression may be possible in the near future.
Similarly, two different methods for construction of the recombinant virus will
be described, because they each have advantages and disadvantages Alternative
methods may be preferable m particular circumstances, and a recent report shows
that recombmation between adenovirus sequences can be accomplished entirely
m E. colt, leading to full-length clones that are infectious upon transfection into
mammalian cells (28). The methods are of course applicable to the construction
of all kinds of adenovirus recombinants.
2. Materials
Young et al.
64
ITR
CMV
1E promoter
and splfctng stgnnls
EcoRV,
Sal1 cloning
sttes
PACE
Adenomms
(21562)
E3 mR.VA 3s
129299)
Fig. 1. Plasmid expression cassettes. (A) PACE contains the left-most 355 bp of
adenovirus type 5 DNA (thick black line) cloned into the EcoRI site of a pBR322derived replicon (thin black line). The adenovirus sequence includes the essential ITR
and packaging signals. This is followed by 0.93 kbp of sequence comprismg the
cytomegalovirus immediate early enhancer and promoter and a chimertc pair of splice
signals (unshaded rectangle). Immediately adjacent to this lies a set of restrictionenzyme cloning sites AgeI, BcfI, EcoRV, and SalI, the latter two being the most useful,
because Age1 is very expensive and BclI is sensitive to dam methylation. The cloning
sites are followed by a sequence containing poly(A) signals (stippled line). Adenovtrus sequence begins again at bp 2966 (bp 1653 of the plasmid) and contmues through
Adenovirus
65
attached. The modified DNA was cloned into the BamHI and SalI sites of
pBR322. Sequence analysis has shown that there is a single 8-bp linker attached
to the final nucleotide of the adenovirus sequence, and that the reported SnuBI
site at bp 35768 is missing in the plasmid (see Note 2). The cDNA of choice is
cloned into the leftwardBa1 site and the unique Not1 site, by directional cloning (see Note 3). As shown in the figure, this arrangement places the cDNA
under the control of the E3 promoter and in close proximity to the spliceacceptor site for most E3 mRNAs (20). The poly(A) signals for the E3A group
of mRNAs are expected to be used.
the EcoRV half site at bp 9197. The complete sequence is available upon request.
(B) The adenovirus type 5 sequences are shown as thick lines, the plasmid sequenceas
a thin line, and the region of Ad sequence that is removed upon cloning as a stippled
box. Basepair numbering 1saccording to the completeAd5 sequence.
Young et al.
66
,7kb,
PACE-derlvatlve
PACE-derivative
8254
1
a-.
LLXI
DNA-PC
Fig. 2. Recombination strategies for creatmg virus vectors from PACE derivatives.
(A). Rescue of the packaging constraint in pJM17. Plasmtd sequences are shown as
checkered lines, the CMV enhancer and promoter and the splicing signals as a thick
black rectangle, the gene to be expressed as a thick stippled lme, and adenovirus
sequences as black lines. The crossovers necessary to create a derivative genome,
capable of being packaged into virus, are depicted as reversed curved lines Neither
plasmid is drawn to scale. Note that in pJM17 the left and right genomic termim are
covalently closed. The 7 kb line above the pBR322 sequence mserted into the unique
XbaI site of d1309refers to the total distance between adenovirus bp 355 and bp 2966
after insertion. Taking mto account the net deletion present in d1309, the extra DNA
that can be accommodated in a stable virus genome, and the net deletion of region E 1
adenovnus sequence in PACE, we estimate that cDNAs up to 3.2 kbp could be incorporated into a recombinant virus, This could be increased considerably by using plasmids similar to pJM17 with extensive deletions in E3. (B) In this diagram, the PACE
derivative DNA is shown linearized for convenience only. Conventions are as for (A),
Adenovirus
67
except that the packaging stgnal (w) is indtcated next to the ITR. LLXl
DNA-PC
digestedwith PueR71and &I gives four fragments,of which only the rightmost one
is shown, with the dotted lme indicating sequence continuing to the right termmus.
Note that PueR71, although an rsoschrzomer ofXho1, does not cleave at the XhoI site
at bp 9699. BecauseLLXl has a net deletion of 1171 bp, the largest cDNA that is
expected to be accommodated in PACE is approx 4.2 kbp.
Young et a/.
68
pPF446-derivative
(uncleaved
DNA)
gene X
/.*+-j
21562
28592
DNA-PC of Ad5 or
Ad5 derivative
(cleaved with EcoRI)
35937
29509
27331
Fig. 3 Recombmatton strategy for creatmg virus vectors from pPF446 dertvatives
Conventions as for Fig. 2B, except that the plasmid is shown as a thm black lure The
size of the DNA that can be accommodated in a repllcatton competent vector IS
expected to be approx 2.7 kbp
SH30072 03) or fetal clone II (cat. no. A-6166-1) (both from Hyclone, Logan,
UT) for A549 and 293 cells respectively, to a final concentration of 10%. Both
the glutamine and the pemcillin and streptomycm mtxture are prepared as 100X
stocks m H20, and are kept frozen at -20C
2 Plaque assay medrum. The abrlity to perform an adequate plaque assay is critical
to creating and analyzing recombinant viruses, The medium used to overlay the
assay plates after infection must be able to keep the cells alive but not actively
dtvtdmg Although several different medta formulattons are useful, the followmg
medium, developed in the laboratory of Harold Ginsberg (29) and referred to as
2xLP, has proved to be extremely rehable wtth a vartety of cell types and mcubation condmons. Medmm 2xLP IS made as follows*
a. Autoclave 2.5 g of lactalbumm hydrolysate (Grbco-BRL, Garthersburg, MD,
cat. no. 670-l 800) and 5 g of Bacto-peptone (Dtfco, Detroit, MI, cat no. 0 11801-8) m 250 mL of detomzed and distilled Hz0 m a 500 mL glass bottle at 120
psi for 20 mm This medium can be made ahead of time and stored mdefimtely
b. After the mix has cooled, add the followmg ingredients directly to the
500-mL bottle
103 mL sterile deionized and distilled H,O
100 rnL stenle 1OX Earles solution from powder (Grbco cat no. 14055-057)
10 mL sterile 50X MEM ammo acids (Gibco cat no 11130-010)
5 mL sterile penicillm and streptomycin solution.
2 mL stertle 100X vitamms (Gibco cat no 11140-050)
Up to 29.6 mL sterile 7 5% solution NaHCOs (Mediatech [Herndon, VA]
cat no 25-035-Ll). The final color should be red, not purple. Ths should be
added last, or a precipitate may form.
Also needed for the plaque assay are supplemental defined calf serum, MgCl*
(30), glutamme, purified agar (BBL cat. no. 11853), and neutral red
69
3. Viral growth medium: Although there are many different media suitable for overlaying infected cells, the following medium, developed by the Ginsberg laboratory (31) and referred to as infecting fluid or IF has proved to be excellent
for growing stocks of virus, analyzing the infectious cycle, and for long-term
storage of unpurified stocks of virus, Even if the stock is frozen and thawed
many times, the titer is maintained at its original level. The formulation and
preparation is as follows:
a. Dissolve 7.4 g of bacto-tryptose phosphate broth (Difco cat. no. 0060-01-6)
in 508.5 mL of deionized, distilled H,O, and autoclave at 120 psi for 30 min.
b. Dissolve 1.4 g of NaHCO, in 250 mL of deionized, distilled H,O and autoclave at 120 psi for 30 min.
c. Immediately before making a batch of IF, combine the two sterile ingredients.
d. Then add: 75 mL of chicken serum (Gibco cat. no. 16110-082; sole supplier),
56.5 mL of concentrated Scherers maintenance medium (BioWhittaker
[Walkersville, MD] cat. no. 06-191B); this must be requested specially, as it is
not made routinely, and 10 mL of stock penicillin and streptomycin solution.
e. Dispense in convenient sizes, typically 100 mL and store at 4C in tightly
capped bottles. A sample should be incubated at 37C for a sterility check.
The shelf life of IF is several months, but non-air-tight bottles may allow it to
become more alkaline with time, and a precipitate may form. (This may happen during the sterility check, and is no cause for alarm.) Although this seems
to have little effect on the replication cycle of the vuus, IF contammg a precipitate should be used for noncritical viral preparations only.
4. Hnt analysis reagents.
a. Nuclear lysis buffer: make a stock of 11.1 mM Tris-HCl, pH 7.4, 11.1 rnA4
NaCl, 11.1 mA4 EDTA, 0.555% SDS. Keep at room temperature. (This stock
can be made easily by diluting the separate ingredients together to give the
appropriate concentrations It will be further diluted by 9/10 with pronase
stock to give a final concentration of 10 mA4TNE, 0.5% SDS.)
b. Pronase stock: Make a stock solution of pronase (Sigma cat. no. 6911) at
10 mg/mL, and incubated at 37C for at least 30 min to digest any contaminating nucleases. Distribute the digested stock in 1.O-mL aliquots and store at
-20C. Tubes can be frozen and thawed several times and the enzyme ~111
retain activity.
5. DNA-PC reagents: The methods for preparing the DNA-PC are a modification of
those described in Chinnadurai et al. (32) and Volkert and Young (33). CsClpurified vinons of LLX 1 are isolated using techniques described elsewhere (34).
There is no need to remove the CsCl by dialysis prior to subsequent steps.
a. Set up a Sepharose CL-4B column as follows: Put 50 mL of Sepharose in a
250-mL Erlenmeyer flask with a suction side-arm. Remove air by applying a
vacuum, holding the flask at an angle at all times. The Sepharose is ready
when it stops bubbling Pipet approx 25 mL of the Sepharose into a small
glass column, such as a 10 x 40 mm disposable column from Bio-Rad. Let the
buffer run through the column into a sterile beaker, and keep reloadmg the
70
Young et al.
b.
c.
d.
e.
g.
h.
1.
buffer onto the column until the matrix is packed. The final packed column
should have approx 1.5 in. of buffer above the matrix within the column m
preparation for loading of the viral band.
Wash the column with 25 mL of newly prepared 4 M guanidine-HCl buffer
(use approx 50 mL to wash a previously prepared column).
Prepare a small volume (approx 1 mL) of approx 8 Mguanidme-HCl
by adding crystals of the solid to a small volume of dtstilled H,O until no more wrll
go into solution. A saturated solution is slightly greater than 8 M.
Dependmg on how much DNA-PC is to be prepared, add an equal volume of
the 8 Mguamdme-HCl
solution to the viral band in a 5-mL snap-cap polystyrene tube In our experience, a sample of 0.2 mL of a very concentrated viral
band will be sufficient to prepare a reasonable concentration of DNA-PC
Vortex briefly. Put the solution on ice for 5-10 min. The translucent viral
band will become clear as the virus structure is disrupted by the guanidine.
Set up the fraction collector to collect I-mL fractions. Add 1 or 2 drops of
0.2% bromphenol blue dye to the disrupted viral solution and load through
the guanidine-HCl buffer on top of the matrix, using a Pasteur pipet. The
column is eluted using a continuous flow of guanidine-HCl buffer. The DNAPC usually emerges at fractions 5-7 and approx 3-4 fractions before those
containing the blue dye. Stop the elution when the dye has reached the bottom
of the column. The column can be reused with another sample of the same
viral preparation, but it must be washed with 50 mL 4 M guanidine-HCI. Do
not let the column dry if it is intended to reuse it.
Keep the fractions on ice and take the A 260/A280value of each fraction using
4 A4 guanidine-HCl buffer as a blank. Select the fractions with the highest AzbO
values for subsequent dialysis. It is important to try to keep the concentration
of DNA-PC as high as possible for the later transfections, so do not pool the
fractions if they have very different AzhOvalues. Peak fractions may have concentrations of 30-50 pg/mL, but those as low as 10 pg/mL are usable.
Dialysis: To promote faithful refolding of the termmal protein, the dialysis is
performed in a number of steps: First, dialyze the fractions agamst approx
100 volumes of 1.3 M guanidine-HCl buffer for 30 min, next, 0.4 M guamdine-HCl buffer for 30 min, and, finally, TE for at least 1 h or overnight,
Place the DNA-PC sample into a sterile Eppendorf tube, and take a final A,&
Asgo reading to calculate the concentration of the DNA-PC. Concentration
changes are minor from the undialyzed sample. The ratio of A260/A280 varies
from approx 1.6-l .9, but all seem to be infectious.
Store the DNA-PC sample on ice in the cold room. It ~111 remam infectious
for many months and despite the apparent lack of sterile technique, it remains
uncontaminated.
4 A4 guanidine-HCl buffer: Dissolve 114.64 g guamdine-HCl from Sigma or
Fisher (Pittsburgh, PA) m 10 rnMTrts-HCl, pH 7.4,l mMEDTA, up to 300 mL.
This will be enough to prepare the columns, elute the DNA-PC, and prepare
the lower concentration buffers for dialysis. It should be kept cold and used
Adenovirus
71
the same day. Do not use old solutions because the guanidine breaks down
quote rapidly.
6. Calcium phosphate transfection reagents.
a Sterile deionized and distilled H,O.
b. Filter-sterilized 10X salmon-sperm DNA as carrier (240 pg/mL).
c 10X Hepes buffer, pH 7 05 (the pH is important)
d. 10X CaCl, (1.25 M).
Keep at 4C.
3. Methods
Young et al.
72
3. After digestion, heat the mix at 65C for 10 min to inactivate the CluI.
4. Remove a small sample of the digested DNA-PC for analysis on an analytical
gel Before running the gel, the sample must be deproteinized with pronase, and
then phenol extracted, ethanol precipitated and resuspended in TE.
5. Store the digested DNA-PC on ice for use up to 24 h later (or at -80C if the
sample is to be used several days later).
3.1 .1.3. PREPARATION OF PACE-DERIVATIVE DNA (SEE NOTE 6)
1. Isolate a pure clone of the bacterium containing the desired plasmid.
2 Prepare the plasmid DNA using proprietary column chromatography (Qtagen,
Chatsworth, CA). We usually use the midiprep kit as thts gives sufficient
DNA for several confirmatory
restriction enzyme digestions and for several transfections.
3.1 .I .4 PRECIPITATION WITH CALCIUM PHOSPHATE (SEE NOTES 7 AND 6)
The precipitation is accomplished by the formation of a calcium phosphateDNA coprecipitate (35). Note: The order of addition of ingredients 1s important.
It is useful to set out a chart for each transfection, in which samples 1 to IE
contain the different plasmids to be used in the creation of the respective
vectors, sample )z+ 1 contams a known plasmid as a positive control for the
production of virus, sample n + 2 contains n.o plasmid as a control for
the efficiency of cutting of the DNA-PC, and sample n + 3 contains uncut
DNA-PC
efficiency.
Total volumes
of the
3.
4.
5
6.
7.
8.
9
10.
11.
73
should be added to the 2xLP stock (see Subheading 2.) and then should be kept
at 37C.
After 4 h, remove medium from a few plates (up to 6 at a time), and add 2 mL
prewarmed glycerol-saline solution (15% glycerol in PBS).
Remove the glycerol-saline 30-60 s after addition. Important. Do not leave on
plates for an extended time, as it is toxic.
Add 4 mL prewarmed Hanks balanced salts solution (10X solution, Gibco cat.
no. 14060-024). Remove.
Add another 4 mL prewarmed Hanks solution, and leave on the dish until all
plates are boosted and washed.
Prepare the overlay medium mix of 2xLP and agar (1: 1) .
Remove Hanks solution, and dtstribute 10 mL of overlay medium per plate.
Let plates solidify >30 min at room temperature and then incubate at the destred
temperature.
Feed the plaque assay at d 6 with 5 mL of fresh overlay medium, and stain it at d 9
with another 4 mL of overlay medium containing neutral red to a final concentration of 0.01%.
Count plaques at d 10 and as long as the stained cells remain viable.
The protocol is essentially the same as for the plaque assay except that
smaller volumes are used for boosting and washing, and the plates have a final
liquid overlay.
1.
2.
3.
4
5.
6
3.1.1.7.
The individual plaques from the direct-plaque assay are potentially a set of
pure clones, whereas the virus pool from the liquid yield is mixed. Provided
background from the latter is low (see Subheading 3.1.2.1.), the products of
the two types of assay can be analyzed in a similar way.
Young et al.
74
3.1.2.1.
CELL CULTURE
Although the original report from McGrory et al. (22) described the use of
direct-plaque assays,we have found the liquid-yield transfection assay, using
35-mm plates of 293 cells, to be reliable and technically easier to perform. This
is because in our hands the large nonviable plasmid pJM17 does not give rise
spontaneously to genomes that are small enough to be within the packaging
limit (breakthrough virus).
3.1.2.2.
(SEE NOTE 9)
Transformation of pJM 17 and other large plasmids mto bacteria often results
in extensive deletion of adenovirus sequences, so great care must be taken to
ensure that the whole genome is still present in the transformant chosen to be
the source of pJM17 DNA.
1. Transform pJMl7 into a suitable bacterial host.
2. Examine plasmid DNA preparations from several transformed colonies, and make
frozen stocks of a correct transformant for future use. Once established, these
transformants appear to be stable. Working preparations can be made by proprietary maxi-prep column chromatography (Qiagen) using 500-mL overnight
cultures of LB broth with 250 &mL of penicillm.
3.1.2.3.
for DNA-PC
transfections
DNAs are substituted for the cut DNA-PC. Ideally, one precipitate should contain JM 17 DNA alone, to check for any breakthrough of infectious vn-us, and a
positive control with a known vector plasmid may be advisable.
1 The amounts of plasmid DNA used should be determined empirically, but we
routinely use 2-5 pg of pJM17 DNA and l-3 pg of PACE derivative DNA.
2. Use 250 pL of calcium-phosphate precipitate per plate.
3. For the glycerol boostuse 1mL of glycerol saline andtwo washeswith 2.5 mL of
Hanks solution.
4. After the secondwash with Hanks, overlay the plates with 3.0 mL of infectmg fluid.
5. Incubate the plates for up to 14 d. Often no CPE is observed at this stage, so the
cells can be frozen away on the plate, transferred to tubes, and fresh plates inoculated with 0.3 mL of the lysate. If the original transfection was successful, CPE
should be observed within 4-5 d after the second mfection. However, we normally keep the plates longer in case the initial transfection sample contains a very
low titer of infectious virus.
should be per-
Adenovirus
75
Viruses
All of the methods for adenovirus vector construction described above have
the potential for yielding either the original virus genome, or some undesired
recombinant structure. Simple methods for genomic analysis of recovered virus
are therefore necessary. We have adapted the classic Hut technique, originally developed for small circular genomes (37), to the larger linear adenovir-us.In most cases,simple restriction digestions of DNA isolated from infected
cells can be used to determine if the virus is the desired recombinant. It is of
course possible to use PCR techniques with ohgonucleotrdes specific for the
junction between viral and the desired gene sequence (38). The modified Hirt
procedure is as follows:
1. Infect a 35-mmdish of the appropriate cell type either with 0.2 mL of the original
transfection yield or with 0.3 mL of an individual plaque isolate.
2 Wait until complete CPE is observed (usually 3-4 d). If CPE is incomplete
by
6-7 d, harvest the yield and perform a secondround of infection using 0.2 mL of
the passage 1 maternal.
3. Remove infected cells to small unsterile snap-cap tube (5-mL polypropylene).
4. Centrifuge cells at -200g for 10 mm at 4C.
5 Resuspend cell pellet in 1 mL of cold PBS and centrifuge again. The pellet can be
stored in a -20% freezer at this stage, if desired.
6 Resuspend m 1 mL of nuclear lysis buffer plus pronase (Subheading 2.4., item 4)
7. Incubate in a water bath at 37C for 15-30 min.
Young et al.
76
8. Add 0.25 mL of 5 A4 NaCl. Mix gently and pour into a 1S-mL Eppendorf tube.
The mixture will be very viscous, and there will be a whitish precipitate.
9 Chill on ice for 30 min or longer; the precipitate can be left indefinitely at 4C
10. Centrifuge at 4C for 5-10 mm.
Il. Remove approx 0 5 mL of clear supernatant and place m an Eppendorf tube. Add
an equal volume of isopropanol to precipitate the DNA. Centrifuge the preclpltate for 15 min. (The remainder of the original NaCl precipitate and supernatant
can be kept m reserve at 4C.)
12. Wash precipitate with 70% ethanol, and then dry the pellet by evaporation.
13. After the DNA is dry, resuspend in 50 pL of TE. This DNA preparation is not
very pure, but it can be digested by most restriction enzymes. If desired, RNase
can be added to a final concentration of 0.5 mg/mL. If the sample IS to be kept for
several days, store tt at -2OC
14. If the DNA samples do not digest well, add 45 ltL of a 1.1 phenol-chloroform/
lsoamyl alcohol mixture to the DNA Vortex and spur for 5 mm at room
temperature.
15. Remove the supernatant very carefully. Be sure not to pick up any of the lower
phenol-chloroform/isoamyl
alcohol phase. This supematant can be used directly
for restriction digestion without a second ethanol precipitation. If the DNA still
does not digest well, precipitate rt a second time with 100% ethanol, wash the
pellet with 70% ethanol, dry, and resuspend as before.
After the restriction analysis (or PCR analysis) has confirmed the expected
genomic organization of the desired vector, it is necessary to purify the virus
by plaque purification.
1. Prepare 60-mm dishes of the appropriate cell type to be Just confluent 2 d
after seeding.
2. Prepare IO-fold serial dilutions of either the original transfection yield from the
liquid yield assay, or of the original plaque isolate from the direct transfection
assay, using IF as the diluent.
3. Perform a plaque assay using several different dilutions of the virus, so that at
least one plate has less than 10 well-isolated plaques. In practice, this is usually obtained by diluting the original plaque isolate 103-, 104-, and 105-fold.
It is harder to Judge the dilutions needed for purification from the liquid yield,
but if CPE was observed on the original transfection plates, use 104-, 105-, and
106-fold dilutions.
4. Aspirate medium from the plates and inoculate with 0.2 mL of the various dilutions
5. Incubate at 37C for 60 mm, rocking the plates to disperse the moculum every
15-20 min.
6. Warm up enough of the 2xLP for all plates (5 mL per plate), add supplemental
defined calf serum to 2%, MgCl, to 5 mM (essential to get large plaques) (30),
7.
8.
9.
10.
11,
12.
13.
14.
15.
16.
77
and glutamine to 0.05%. Keep the medium at 37C for 15 mm to 30 min, but no
longer, as a precipitate may form.
Separately, make up a solution of purified agar to 1.6% in deionized, distilled H20.
Autoclave at 120 psi for 20 min, and keep molten at 5OC.
Immediately before overlaying dishes, mix 2xLP stock and supplements and then
with agar in a 1: 1 mix.
Distribute 10 mL per dish.
After the agar has solidified completely (30-40 min at room temperature), incubate at 37C.
Feed plates on d 5 using 5 mL of agar/2xLP mix per dish. Feed again if the dishes
become acidic (yellowish).
To stain plates use 4-5 mL of mix with neutral red to 0.01% concentration per dish.
Isolate virus from well-separated plaques from the plate showing the least number of plaques, by using a plugged sterile Pasteur pipet to aspirate the infected
cells, which are then placed m 1 mL of IF in a sterile 5-mL tube
Freeze and thaw the samples two to three times to release intracellular virus, and
repeat the plaque purification (see Note 10).
The plaque isolates can be stored at -20C for many years with no loss of titer
We normally isolate three to five individual plaques for safe-keeping, but proceed to detailed analysis with only one.
3.4. Preparation
78
Young et al.
10. If desired, large stacks can be prepared from passage 2 for subsequent purification by CsCl-gradient centrifugation, as described m detail previously (34).
Assay
Unlike the plaque assay, which takes up to 2 wk to complete, the fluorescent-focus assay (FFA) gives results within 2 d. The dtsadvantages are that
rt requires more hands-on time, more expensive reagents, and a UV microscope. In our experience the titers derived from the FFA and plaque assay
are equivalent.
1. Prepare 35-mm dishes of the appropriate cell type, to be confluent m 2 d.
2. Make serial IO-fold dilutions of the viral stock, using IF.
3. Aspirate the medmm and inoculate the dishes with 0.2 mL of the diluted vu-us
stock. Because this 1s a microscopic assay, and mdividual infected cells are
scored, the diluttons to be inoculated are usually those of 104, 1OS,and 1O6
4. Incubate the infected plates at 37C for 24-28 h
5 Aspirate the medium, wash two times with 2.5 mL of PBS, and then remove the
second wash and add 2.5 mL of 90% methanol. Note* Leave at room temperature
for at least 4 min. This fixes the cells and the monolayer becomes opaque.
6. Remove the methanol, and wash twice with 2.5 mL of PBS, lettmg the PBS
remain on the cells for at least 4 min each time. This rehydrates the cells The
fixed plates can be stored for several days under PBS at 4C
7. Remove the PBS, and add 0.5 mL of diluted rabbit antiadenovirus antiserum.
Polyclonal antiserum directed against purified virions can be prepared in large
quantities, and is stable for many years at -2OC. The dilution required is tested
empirically, but typical dilutions are 1 200 in PBS Incubate at room temperature
for 30 min. Wash twice with 2.5 mL of PBS.
8. Add 0.5 mL of a solution containing fluoresceinated goat antirabbit antiserum
(Boehringer Mannheim, Indianapolis, IN, cat. no. 605 210) and rhodamine-conjugated BSA (BBL, cat no. 40824), and incubate for 30 min. The dilutions of
both conjugates are usually made 1.40 to 1:50 in PBS, and the solution IS kept
frozen, and m the dark.
9. Remove the stain, wash twice with 2.5 mL of PBS, add 0.5 mL of PBS to keep
the dishes moist, and examine under a UV-fluorescence microscope. Dishes can
be stored at 4C, but must be kept dark to prevent bleaching of the fluorescem.
10. Count the number of fluorescing cells per optical field, whose sizedependson
the properties of the microscope. Count a sufficient number of fields to give at
least 100 fluorescent foci, and calculate the average number of such foci per
79
4. Notes
1. The only useful cloning sites in the original PACE plasmid are the blunt EcoRV
and 5 cohesive sun sites, In part this stems from the way m which the various
modules of the promoter, enhancer, splicing signals, and polyadenylation signals were assembled in the precursor plasmid and m part from the large number
of restriction sites present in the adenovirus sequences from bp 2966 to bp 9 197.
2. Both restriction enzyme and sequence analysis of pPF446 show that the SnuBI
site reported in the complete Ad5 sequence in the database (GenBank Accession
no. M73260) is missing m the plasmid. The sequence in our version of Ad5 is
TACGTC, the same sequence as in Ad2. We assume, but have not proved, that
this is a naturally occurrmg restriction enzyme polymorphism.
3. The two %a1 sites in pPF446 are differentially cleaved. The rightward one (Ad5
bp 30470) is usually resistant to cleavage if the plasmid is propagated in a dam+
strain of bacterium, presumably because the methylated A residue immediately
adjacent to the %a1 site interferes with site recognition by the enzyme
4 In the original paper by McGrory et al. (22), direct transfection of pJM 17 DNA
into 293 cells gave rise to plaques late in the assay. Analysis of the genomes
showed that they contained deletions that resulted in sizes below the packaging
hmit. We have seen no evidence of such deleted genomes in the liquid yield
assays described in Subheading 3.1.2.
5. The origins of virus LLXI were outlined in Brunet et al. (24). Subsequent analysis of junctions between the Ad5 and Ad22+ND1 sequences has revealed a complex chimera, as follows:
Ad5 from the left-hand end to bp 1475.
No sequence differences between Ad5 and Ad2 from bp 1476 to bp 1500.
Ad2 from bp 1501 to bp 3609.
There is a single base addition from bp 3610 to bp 4059 (A at bp 4040 in Ad5).
Ad5 from bp 4060 to bp 7879.
There are two sequence differences between Ad5 and Ad2 from bp 7880 to bp
826 1, and the precise sequence is not yet known m this area.
Ad2 from bp 8262 to bp 155 17
No sequence differences between Ad2 and Ad5 from bp I55 18 to bp 15605,
Ad5 from bp 15606 to bp 27643
No sequence differences from bp 27644 to bp 27648.
Ad2 bp 27649 to the right hand end.
It is important to point out that the right hand end contains part of the SV40 T
antigen gene embedded in E3 (25).
The total genome size is 34766 bp in length.
6. As is well-known, transfections work best if close attention is paid to the mamtenance and preparation of the cells used. Human 293 cells, which are used for the
transfections involvmg PACE, should be used at low passage number, and the
80
8.
9.
10.
11.
Young et al.
plates to be transfected should be just confluent. Cells that have been at confluence
for some time do not transfect efftctently, and underconfluent monolayers are easily disturbed by the manipulations of the transfection, and also may not survtve
under the agar overlay. The stock cultures are maintained in DME with 10% fetal
clone II (Hyclone), and are split once a week, at a dilution of 1:20. Removal of the
cells from the culture flask is accompltshed by standard techniques, namely washing with PBS, and brief treatment with trypsin-EDTA. In our experience, it is not
desirable to ttturate the cells extensively to make a single-cell suspension, and thus
we do not count the number of cells removed from the flask. A T75 flask will yield
about thirty 35-mm or fifteen 60-mm dishes ready to transfect in 2 d. One-day-old
plates are not used, because the monolayers are not stable to manipulation
The preparation of plasmid DNA for the overlap reaction has been performed in many
different ways, including the use of crude minipreps produced by the boilmg lysts
method (40). Recently Qiagen has repotted that the presence of LPS in some plasrmd
DNA preparations mhtbits the efficiency of transfection. We have not determined if
this is a problem m adenovirus-overlap recombmatton, but caution may be m order.
Although several alternative proprietary preparations for transfectton using ltposome micelles have been developed, we have not found them to be sigmficantly
better than the calcium-phosphate method first described by Graham and van der
Eb (35). The latter is reliable and cost-effective. We have not tested electroporation as a method for vnus reconstruction.
Different methods for preparing pJMl7 have not been evaluated by us, and
because the efficiency of recombination with PACE derivatives is low, tt may be
worthwhile to see if CsCl-purified preparations are more efficient.
Although plaque-purification
is time-consummg, it 1snecessary because the original transfection may yield a mixture of genotypes, and even if there is a smgle
genotype, the population is not clonal. At least one round of plaque purificatton
is necessary, and many investigators have suggested three rounds of purification.
It should also be emphasized that if a set of different recombinants has been
created in a particular transfection, the plaque-purificatton stage must be conducted under conditions in which crosscontamination
cannot occur. NoteRestriction-enzyme analysis and, if necessary, sequence analysis is performed on
Hirt DNA derived from cells infected with samples of the final plaque Isolate to
confirm the identity of the recombinant,
If several different recombinants have been created in a transfection, each one is
handled separately durmg the preparation of the working stocks. We inoculate
the T25 flasks at different times, and designate samples of IF for each stock.
Minor crosscontaminatton
between isolates from aerosols created durmg
maculation would be possible if suitable precautions are not taken, and likely to
escape notice.
References
1. Berkner, K. L. (1988) Development of adenovirus vectors for the expression of
heterologous genes. BioTechniques f&616-629.
81
2. Graham, F. L. (1990) Adenovnuses as expression vectors and recombinant vaccines. TIBTECH 8, 85-87
3. Berkner, K. L. (1992) Expression of heterologous sequences in adenoviral vectors Curr Topxs Microblol Immunol. 158,39-66.
4. Gluzman, Y., Reichl, H., and Solnick, D. (1982) Helper-free adenovirus type 5
vectors, Eukaryotlc viral vectors (Gluzman, Y., ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 187-192.
5. Schwarz, J. K., Bassing, C. H., Kovesdi, I., Datto, M. B., Blazing, M., George, S.,
Wang, X.-F., and Nevins, J. R. (1995) Expression of the E2Fl transcriptton factor
overcomes type p transforming growth factor-mediated growth suppression. Proc.
Natl. Acad. SCL USA 92,483+87.
6. Liu, T.-J., Zhang, W.-W., Taylor, D. L., Roth, J. A., Goepfert, H., and Clayman,
G. L. (1994) Growth suppression of human head and neck cancer cells by the
introduction of a wild-type pS3 gene via a recombinant adenovirus. Cancer Res.
54,3662-3667.
7. Wills, K. N., Maneval, D. C , Menzel, P., Harris, M. P., Sutjipto, S , Vaillancourt,
9.
10
11.
12.
M.-T., Huang, W.-M., Johnson, D. E., Anderson, S. C., Wen, S. F., Bookstem, R.,
Shepard, H. M., and Gregory, R. J. (1994) Development and characterizatton of
recombmant adenoviruses encoding human p53 for gene therapy of cancer.
Human Gene Ther 5, 1079-1088.
Mul, Y M., Verrijzer, C. P., and Van der Vliet, P C. (1990) Transcription factors
NFI and NFIII/oct-1 function independently, employing different mechanisms to
enhance adenovn-us DNA replication. J Vu-01. 64,5510-5518.
Coenjaerts, F. E. J. and Van der Vliet, P. C. (1995) Adenovirus DNA rephcation
in a reconstituted system. Methods Enzymol 262,548-560.
VerrtJzer, C. P , Kal, A. J , and Van der Vliet, P. C. (1990) The DNA binding
domain (POU domain) of transcription factor act-1 suffices for stimulation of
DNA replication. EMBO J. 9, 1883-1888.
Bennett, C. B. and Rainbow, A. J. (1989) DNA damage and biological expression
of adenovirus. a comparison of liquid versus frozen conditions of exposure to
gamma rays Radzat Res. 120, 102-l 12.
Arnold, W. R. G. and Rainbow, A. J. (1996) Host cell reactivation of irradiated
adenovirus in UV-sensitive Chinese hamster ovary cell mutants. Mutageneszs 11,
89-94.
13. Colicos, M. A., Haj-Ahmad, Y., Valerie, K., Henderson, E. E., and Rainbow, A. J
(199 1) Construction of a recombinant adenovirus contaming the den V gene from
bacteriophage T4 which can partially restore the DNA repair deficiency m xeroderma pigmentosum fibroblasts. Carcznogenesis 12,249-255.
14. Young, C. S H. (1995) Homologous recombinatton m the replicative cycle of
adenoviruses and its relationship to DNA replication. Curr. Topics Mcroblol
Immunol. 199/11, 89-108
15. Park, M. S., Ludwig, D. L , Stigger, E., and Lee, S. H. (1996) Physical mteraction
between human RAD52 and RPA is required for homologous recombination m
mammalian cells. J Blol. Chem. 271, 18,99619,000.
Young et al.
82
21 Bett, A. J., Haddara, W., Prevec, L., and Graham, F. L. (1994) An efficient and
flexible system for construction of adenovirus vectors with insertions or deletions
m early regions 1 and 3. Proc. Natl. Acad Sci USA 91,8802-8806.
22 McGrory, W. J., Bautista, D S., and Graham, F. L (1988) A simple technique for
the rescue of early region 1 mutations into infectious human adenovirus type 5.
Virology 163,614-617.
23. Bett, A J., Prevec, L., and Graham, F. L. (1993) Packaging capacity and stability
of human adenovirus type 5 vectors. J Virol 67,59 1 l-592 1.
24. Brunet, L. J., Babiss, L. E., Young, C. S. H., and Mills, D. R. (1987) Mutations m
the adenovirus major late promoter: effects upon viability and transcription during infection. Mol Cell Bioi. 7, 1091-l 100.
25. Kelly, T. J. and Lewis, A. M. (1973) Use of non-defective adenovirus-simian
virus 40 hybrids for mapping the simian virus 40 genome. J. Vwol. 12, 643-652
26. Graham, F. L , Smiley, J., Russell, W C., and Nairn, R. (1977) Characteristics of
a human cell line transformed by DNA from human adenovirus type 5 J. Gen
Vzrol. 36,59-72.
27. DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P.-M., Miller, J. H., and Calos, M.
P. (1987) Analysis of mutation m human cells by using an Epstein-Barr virus
shuttle system. Mol. Cell Biol 7, 379-387.
28 Glard, D. J., Aaronson, S. A., Todaro, G. J., Arnstein, P., Kersey, J. H,, Doslk, H ,
and Parks, W. P. (1973) In vitro cultivation of human tumors. establishment
of cell lmes derived from a series of solid tumors J Nat/ Cancer lnst 51,
1417-1423.
29. Lawrence, W. C and Ginsberg, H. S (1967) Intracellular uncoatmg of type 5
adenovirus deoxyribonucleic acid. J Virol. 1,85 l-867.
30. Williams, J. F. (1970) Enhancement of adenovlrus plaque formation on HeLa cells
by magnesium chloride. J, Gen. Virol. 9,25 1-256.
Adenovirus
83
3 1. Ginsberg, H. S., Gold, E., and Jordan, W. S. (1955) Tryptose phosphate broth as
supplementary factor for maintenance of HeLa cell tissue cultures. Proc. Sot.
Exp. Biol. Med 89,66-71.
32. Chinnadurai, G., Chinnadurai, S., and Green, M. (1978) Enhanced mfectivrty of
adenovirus type 2 DNA and a DNA-protein complex. J. Virol. 26, 195-199.
33. Volkert, F. C. and Young, C. S. H. (1983) The genetic analysis of recombmation
using adenovirus overlappmg terminal DNA fragments. Gology 125, 175-l 93.
34. Green, M. and Wold, W. S. M. (1979) Preparation of human adenoviruses: growth,
purification, and transfection assay. Methods Enzymol. 58,425-435.
35. Graham, F. L. and Van der Eb, A. J. (1973) A new technique for the assay of
infectivity of human adenovirus 5 DNA. Virology 52,456-467.
36. Frost, E. and Williams, J. (1978) Mapping temperature-sensitive and host range
mutattons of adenovuus type 5 by marker rescue. Virology 91,39-50.
37. Hut, B. (1967) Selective extraction of polyoma DNA from infected mouse cell
cultures. J. Mol Bzol. 26, 365-369.
38. Zhang, W.-W., Fang, X., Branch, C. D., Mazur, W., French, B. A., and Roth, J. A.
(1993) Generation and identification of recombinant adenovirus by liposomemediated transfection and PCR analysts, BloTechnzques15,868-872.
39. Philipson, L. (1961) Adenovirus assay by fluorescent cell-counting procedure.
Virology 15,263-268.
40 Holmes, D. S. and Quigley, M. (198 1) A rapid boiling method for the preparation
of bacterial plasmids. Anal. Biochem. 114, 193-197.
7
Construction
of Mouse AdenovirusType
1 Mutants
85
and Protocols
NJ
86
2. Materials
1. 5MNaCl.
2. 50% Polyethylene glycol (PEG8000, Sigma, St. Louis, MO, cat. no. P-2139):
Weigh out 250 g of PEG and add water to 500 mL. Stir until dissolved, and
Construction of MA V- 1 Mutants
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
2 1.
22.
23.
24
25.
26.
27.
28.
29.
30.
3 1.
32.
87
88
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
Construction of MA V- 1 Mutants
89
63. Wizard DNA Clean-up Kit (Promega, Madison, WI, cat. no. 47280). Store at
room temperature.
64. fmol Sequencing Kit (Promega cat. no. 44110). Store at -20C
65. ym3*PdATP 3000 Ct/mmol. Store at -20C.
66. 8% Urea, 6% polyacrylamide gel: 5 mL 10X TBE, 7.5 mL 38:2 acrylamide, 18.3 mL
water, and 24 g urea. Stir for at least 15 min. Add 300 pL of 10% ammonium
persulfate and 20 pL of TEMED and cast immediately.
67. Sequencing gel fix: 10% methanol/lo% acetic acid. Store at room temperature in
a brown bottle.
68. 35-mm Polystyrene tissue-culture plates (Corning cat. nos. 25000-35 or 258 10).
69. loo-mm Polystyrene tissue-culture plates (Corning cat. no. 25020).
70. 150~mm Polystyrene tissue-culture plates (Corning cat. no. 25030- 150).
71. Small glass test tubes with caps.
72. 1 2% Agarose: Weigh agarose (low EEO), add water, and autoclave. Store at
room temperature.
73. 2X DMEM. For 2 L add 53.5 g DMEM powder (Gibco-BRL cat. no. 12 lOO-012),
14.8 g NaHCO,. Star for at least 4 h and pH to 7.2 with HCl. Brmg volume to
1700 mL. Filter sterilize with 0.2-p filter to 425-mL aliquots. Incubate one
bottle at 37C overnight to check for contamination. Per 425 mL of 2X DMEM,
add 10 mL 100X glutamine, 10 mL 100X pen/strep, 10 mL 100X nonessential
ammo acids, 25 mL 1 MMgCl,, and 20 mL HICS. When these components are
added, the solution IS 2X. Add glutamine to 2X every 2 wk. Store at 4OC. Do not
use if >2 mo old.
74. 100X Nonessential amino actds (Gibco-BRL cat. no. 11140-019).
75. 1 M MgC12. Autoclave and store at room temperature. Use only for cell culture.
76. 100X Neutral red: 1% neutral red in water. Filter through Whatman no. 1, boll,
then through 0.2~pm filter. Store at room temperature.
77 TE-equilibrated phenol (33)
78. Chloroform
79. Oligonucleotide-directed
mutagenesis kit (Amersham, Arlington Heights, IL, or
Stratagene).
80. Plugged pipet tips.
8 1. StrataClean resin (Stratagene).
3. Methods
3.1. Preparation
of DNA-Protein
Complex
90
Construction of MA V- 1 Mutants
91
2. Close the outlet valve on the gradient maker and add 5 mL of 1.2 g/cm3 CsCl and
a stir bar to the chamber proximal to the outlet valve. Open the valve between the
two chambers and allow a small amount of the liquid to flow into the distal chamber to remove the air from the valve. Close the valve and pipet the liquid from the
distal chamber to the proximal chamber. Add 5 mL of 1.4 g/cm3 CsCl to the
distal chamber.
3. Place the tubing into the bottom of a 13-mL ultracentrifuge tube. Start the stirrer,
then open both valves and turn on the peristaltic pump simultaneously. Fill the
tube from the bottom and monitor the progress of the gradient so that no bubbles
are released as the gradient is completed. Gently remove the capillary tube from
the solution by sliding it up one wall of the tube so as not to disturb the gradient.
Make four to stx gradients for this procedure.
4. Load the diluted virus from Subheading 3.1.2., step 8 to the top of the gradients.
As necessary, add 10 mMTris-HCl,
pH 8.0, to fill tubes to within a few millimeters of the top of the tube.
5. Centrifuge at 35,000 rpm (210,OOOg) m an SW41 swinging bucket rotor at 4C
for at least 16 h (see Note 4).
6. In the second gradient a major band should be seen, with possibly a faint band
above it Using the dripper as described above, collect the bottom white band,
which contains vuus. Discard the rest of the gradient, including the top white
band containing mostly cellular debris and top components.
7. Dialyze the samples for 2 h to overnight against 10 mMTris-HCl,
pH 8.0, at 4C
m 12,000-14,000 molecular-weight exclusion-dialysis tubing (see Note 6).
8 Place no more than 3.5 mL of sample m a conical polypropylene 15-mL tube
with graduations and add the following: 70 pL 1 MTris-HCl, pH 8.0, 14 pL 0 5 M
EDTA, 3.5 mL 8 Mguamdme HCl, 3.57 g optical grade CsCl, and dH20 to a
final volume of 7 mL (see Note 7).
9. Mtx well by mverston and pour into 13-mL ultracentrifuge tubes. Fill to within a
few millimeters of the top with mineral oil.
10. Centrifuge m an SW4 1 swinging bucket rotor at 28,000 rpm (134,000g) for 40 h
at 15C (see Note 4).
11. Using the dripper and the peristaltic pump as described above, fractionate the
gradient into 0.5~mL ahquots (approx 20-25 drops per fraction), collecting 15-20
fractions per gradient in capless microfuge tubes.
12. Measure the AX6s of each fraction (dilute fractions in TE if necessary) and plot.
Pool the fractions that form the peak.
13. Dialyze in 12,000- 14,000 molecular-weight exclusion-dialysis tubing against 2 L
of chtlled TE at 4C for four changes of at least 4 h each.
14. Measure &s and calculate the concentration using the following formula: Q&,)
(50 pg/mL) (dilution factor)/1000 = concentration in pg/pL. The total yield
should be 100-200 pg.
15. Store m microfuge tubes in 5- or lo-pg aliquots Freeze quickly in dry ice or
liquid nitrogen and store at -70C.
92
3.1.4. Preparing DNA-Protein
1. Thaw 5 pg of DNA-protein complex (Subheading 3.1.3., step 15) per transfection sample and digest it with 8 @., ofEcoRI (20 VI&) in a final concentration of
1X ,??coRI buffer containing 1 mMDTT in a final volume of 250 pL. Digest 4 h to
overnight at 37C. (See Subheading 3.2. to determine the number of aliquots of
DNA-protein complex needed.)
2. Partially fill in the sticky ends created by the EcoRI using 8 U of Klenow polymerase and a final concentration of 1 mM dATP. Incubate for 30 mm at 37C.
Stop the reaction by adding EDTA to a final concentration of 25 mA4. Store at
-20C until ready to use
3. Linearize 4 pg of plasmid containing the desired MAV-1 mutation(s) by cuttmg
at a unique site within the vector. Store at -2OC until ready to use (see Note 8).
93
Table 1
Transfection Experimental Plan
Tube
no.
1
2
3
4
DNA-protein complex
PL
I43
5 (digested,
290 5
filled in)
5 (digested,
290.5
filled in)
5
290.5
(undigested)
-
Mutant plasmid
I%
clt
10
4
2.5 MCaCI,,
CiL
50
Hz0 to
500 pL
138.5
15
50
144.5
15
50
144.5
20
50
430.0
7. Set up a DNA mix for each transfection in a 1.5-mL microfuge tube as in Table
1. Using salmon-sperm DNA (1 pg/pL), adJust the total DNA to 20 pg and using
sterile water adjust the final volume to 500 pL. Numbers are as follows* 1,
experimental sample; 2, background religation control; 3, positive control, 4,
negative control.
8. Vortex each of the transfection mix tubes and proceed munediately to the subsequent steps.
9. Aspirate media from two to four plates at a time, and add 1.5 mL of warm 1X
HEBS, pH 7.03 f 0.05 to each plate. Number the plates as above, remembering
that the calcium phosphate precipitate will be split between two plates, generatmg plates 1A and lB, and so on.
10. Using a l&L disposable plugged polystyrene pipet, add the DNA mix for the
first sample to the polystyrene tube (prepared in step 6). Do this slowly over
approx 45 s, while gently flicking (see Note 11). As soon as a precipitate is seen,
add half of the mix to each of the appropriately labeled plates containing 1.5 mL
of 1X HEBS, pH 7.03 f 0.05 (prepared in step 9). Start a stopwatch and note the
time. Let the plates stt for 20 min at room temperature.
11. Repeat steps 9 and 10 for the rest of the samples, allowing 3-5 min between
each sample.
12. At the end of the 20-min incubation, add 3 mL warmed 1X DMEM containing
5% HICS. Check each plate under the microscope for a sandy, grainy appearance
of the precipitate. (If the precipitate appears clumpy, the transfection may not
work.) Incubate at 37C.
13. After incubating for 3-4 h, glycerol shock the cells. Remove the media from two
plates at a time. Add 1.5 mL of warmed 15% glycerol in 1X DMEM and let it sit
for exactly 60 s (use a stopwatch). Aspirate the media containing glycerol and
wash carefully with 3-5 mL warmed 1X PBS. Aspirate the PBS and add 5 mL 1X
DMEM containing 2% NBCS after infecting. Incubate overnight at 37C.
14. After completing the transfection, infect the remaining cells for a plaque assay
with the parent virus from which the DNA-protein complex was made. Follow
94
the directions in Subheading 3.5. to infect the cells for a plaque assay, but
instead of overlaying with agarose, add 5 ml/plate of 1X DMEM containing 2%
NBCS. Incubate overnight at 37C.
15. The next day, aspirate the media from one set of the transfection plates from step
13 (i.e., lA, 2A, and so on) and overlay with 5 mL of media/agarose as in Subheading 3.5. Change the media on the duplicate plates (lB, 2B, and so on) to
fresh 1X DMEM containing 5% HICS. Overlay all of the plates for the virus
infection (plaque assay control) from step 14. Follow the directions for overlaying and checking for plaques as m Subheading 3.5.
of Mutants
Construction of MA V- 1 Mutants
3.
4.
5.
6.
7.
8.
9
10.
11.
12.
13.
14.
95
monolayer once with IX PBS. Scrape cells with a rubber pohcman in 1X PBS
and transfer the solution to a 13-mL polypropylene tube. Centrifuge at 200&
3000g for 10 min at 4C.
Aspirate the supernatant without disturbing the cell pellet. (The cell pellets can
be frozen at -2OOC if necessary.)
Resuspend each pellet in 0.5 mL Hirt solution 1 and vortex very gently to resuspend the cells.
While gently vortexing, add 0.5 mL Hirt solution 2 to which 0.1 volume of
20 mg/mL pronase has been added.
Cap the tubes and incubate at 37C for 2 h.
Add 0.25 mL of 5 M NaCl to each sample and flick the bottom of each tube to
mix. Incubate overnight on ice at 4C
Centrifuge at 17,000g for 45 min at 4OC.
Transfer supematant to a 4-mL polypropylene tube and extract twice with an
equal volume of phenol.
Add 2 5 volumes of ethanol to each tube and incubate on ice or at -2OOC for 15 min.
Centrifuge at 10,OOOg for 15 min at 4OC. Discard the supematant. (No salt is
needed to precipitate the DNA since the supematant contains 1 M NaCI.)
Resuspend pellet m 0.3 M NaOAc, pH 6.0, and transfer to a 1.5-mL microfuge
tube. Add 1 mL 95% ethanol to tube and precipitate as above.
Wash pellet with 1 mL 70% ethanol and centrifuge for 5 min.
Air-dry the pellet and resuspend m 50 pL of water or TE.
Use l-5 $ in PCR or 10 @+per restriction enzyme digest and electrophorese on
a 0.7% agarose gel. If usmg m a restriction digest, also add 1 pg RNase per
sample. Include appropriate control DNA samples in analysis.
96
2. If the mutation results in a stgnificantly smaller PCR product than that of wlldtype (Le., the mutation 1s a deletion), the completed PCR reactions may be simply electrophoresed on a 7% native polyacrylamlde gel after loading dye has
been added (5% glycerol final concentration) (1). Electrophorese at 200 V for 2 h.
Stain with ethidium bromide for 1O-20 min and destain with water for 1O-20 mm.
View and photograph on a UV transilluminator.
3. If a restriction site that is unique to the PCR product has been incorporated mto
the mutation, the PCR product can be digested with the appropriate enzyme to
determine if the DNA 1smutated (1). In this case, aliquot half of each completed
PCR reaction to mrcrofuge tubes and digest with the appropriate enzyme. Electrophorese the undigested and digested samples using electrophoresis and detection conditions as above.
4. Point mutations may also be screened using differential PCR (2). In this case,
two primers are designed that will differentially amplify the wild-type and
mutant DNAs. This can be accomplished by havmg the 3 nucleotlde in each
oligonucleotide correspond to the wild-type or mutant sequence. The PCR conditions to differentially amplify mutant and wild-type products will have to be
empirically determined. To do this, try varying the Mg* concentration and/or
the annealing temperature. The PCR products are then electrophoresed and
detected as described.
5. If the mutation does not meet the above criteria, the PCR product can be
sequenced. After the PCR reaction 1scomplete, remove the excess primers usmg
the Wizard DNA Clean-up Kit (Promega). Analyze 5-10 & of the cleaned PCR
product by electrophoresls to ensure that the primers have been removed and that
the expected PCR product was produced. Use 5 pL of the purified PCR product
as template in sequencing reactions, following the directions given m Promegas
fmol Sequencing Kit. The best results are obtained using end-labeled 32P-oligonucleotides as the sequencing primers. Using the bromophenol blue and xylene
cyan01 dyes as markers for oligonucleotide
migration, electrophorese the
sequencing reactions on an 8% urea, 6% polyacrylamide gel until the area of
interest is in a readable position on the gel. Fix the gel in 10% methanol/lo%
acetic acid for 30-45 min and rinse in water briefly. Dry on a gel dryer and
expose to film overnight. Read the film after exposure to determine if the desired
mutations have been incorporated mto the viral DNA.
Construction of MA V- 1 Mutants
97
98
3.5. Quantitation
1. For each mutant, set up thirteen 60-mm plates of 3T6 cells to be approx 70%
confluent at the time of infection.
2. For each mutant, aliquot exactly 0.9 mL PBS to seven small glass test tubes with
caps and number and order these tubes from 2 to 8.
3. Warm to 37C the I-mL aliquot of each virus stock to be titered (Subheading
3.4.2., step 2). Do not leave at 37C for prolonged periods. Once the stock is
thawed, store on ice.
4. Aliquot exactly 0.1 mL of virus into tube 2. Vortex and remove exactly 0.1 mL
from tube 2 and deposit it into tube 3. Vortex and repeat for the remaining tubes,
creating a dilution series of the original virus stock. Repeat for each virus stock.
5. Aspirate all but 0.5-l mL of media from 13 plates. Label two plates per dilution ( 103-108) and one plate as mock. Add 0.1 mL of PBS to the mock plate.
6. Beginning with the highest tube number (8), the most dilute virus sample, add
exactly 0.1 mL of the solution to each of the two plates labeled 108. Rock the
plates to cover the cells with the liquid. Repeat sequentially in descending order,
but omitting tube 2, as there will be too many plaques to count from this dilution
(see Note 18).
7. Incubate at 37C for 1 h. Meanwhile, microwave sterile 1.2% agarose until it is
boiling and place it in a 45C water bath. Allow agarose to equilibrate at 45C for
at least 30 min. (You will need 2.5 mL per plate, but allow some extra.) Warm 2X
DMEM containing 4% HICS that is supplemented with glutamine, pen/strep, and
nonessential amino acids all at a final concentration of 2X, as well as MgC12 that
is at a final concentration of 50 mM (see Note 19). (You will need 2.5 mL per
plate, but allow some extra.)
8. After the 1 h incubation, mix equal volumes of warm 2X DMEM and agarose.
Working quickly, overlay each plate with 5 mL of medialagarose by gently
applying the solution to the cells with a wide-mouth pipet, touching the pipet tip
to the inner wall of the plate. Try not to introduce bubbles onto the plates. Allow
to stand until the leftover media/agarose in the bottle has set, approx 3-5 min.
Place the plates in the 37C incubator.
9. The next day, and every second or third day thereafter, overlay each plate as
described above with 2 mL per plate, i.e., 1 mL of 2X DMEM mixed with 1 mL
of agarose.
10. When plaques become visible (d 5-9), add neutral red to a final concentration of
1X to the media each time the plates are overlaid. Begin counting and recording
the number of plaques when they become visible and continue counting and overlaying until no or only a few new plaques appear for l-2 d (see Note 20). Always
count the plaques prior to overlaying since the recent addition of media/agarose
tends to make the plaques very hard to see.
11. When no or very few new plaques appear for l-2 d, total the number of plaques
for each plate and multiply that number by the dilution factor. This number is the
titer of the virus in PFU/mL. The most accurate titer will be determined from at
least two plates with 10-100 plaques per plate. For example, if two lo6 plates
Construction of MA V- 1 Mutants
99
4. Notes
1. pmE30 1 and pmE 10 1 are MAV- 1 wild-type variants that contain only one EcoRI
site in the genome. pmE301 contains a single EcoRI site in the ElA region,
whereas pmElO1 contains a single site in the E3 region (1,2).
2. 1X DMEM contains 0.15 A4 salt and because the volume of NaCl to be added is
not negligible, be sure to include it in the calculation.
3. Keep movement of the gradient tubes to a minimum to avoid disturbing the gradients and make sure that a layer is visible in each one prior to loading your
samples, If no layer is present, discard and make another gradient.
4. Because these are equilibrium density gradients, the centrifugation can go longer
than the indicated time.
5. We use a Hoeffer dripper: Follow the manufacturers instructions for your dripper. Using the dripper can be tricky, so it is advisable to practice the procedure on
a blank tube first. This allows you to work out the best way to puncture the tube
and to control the flow rate. We also recommend cleaning the dripper needle
after each gradient to prevent clogging. To clean, insert a needle through
the opening a few times to free any plastic. The dripper needle should be washed
by injecting ethanol and/or water into the opening with a syringe. We recommended cleaning the needle after every sample or every other sample and when
you finish a procedure.
6. Purified MAV-1 DNA can be made by digesting the dialyzed virus in 0.5% SDS
and 250 pg/mL of proteinase K at 50-55OC for 15 min. Extract with phenol one
to two times and re-extract the first phenol phase with TE. Extract with chloroform twice to remove the phenol. Dialyze against TE, pH 8.0, at 4C for four
changes of at least 4 h each. Read the AZ60and store at 4C. Expect the yield to be
10.1 mg/mL.
7. When making the gradient containing guanidine HCl, put the dialyzed virus onto
two gradients to have a balance in the centrifuge.
8. To generate a plasmid with a mutation in an MAV-1 gene, first clone the wildtype gene of interest into a vector and mutagenize using oligonucleotide-directed
mutagenesis (Amersham or Stratagene), PCR mutagenesis, or by replacing
genomic DNA sequence with cDNA sequence. To screen for potential mutants
generated by oligonucleotide-directed
mutagenesis, a restriction site unique to
the gene region to be mutagenized can be incorporated into the mutation site. The
presence of the site will distinguish mutant from wild-type in subsequent steps.
To prevent digestion of the plasmid DNA by the enzyme used to digest DNAprotein complex (when the two are mixed together for the transfection), you can
first treat the plasmid DNA with the appropriate methylase (EcoRI methylase in
this case), following the manufacturers instructions (2).
100
0.1 mL of this will be used to inoculate the plate, thus it is a loo-fold (1OX)
dilution; the D.O.P. will be lo*.
101
19. When overlaymg cells with medialagarose for a plaque assay, keep the media
and agarose separate until ready to overlay. Warm the media in a container
that is big enough to accommodate the agarose as well. When ready to overlay, add an equal volume of agarose to the aliquot of media and use immediately, Have separate aliquots of media/agarose
for each mutant when
overlaying and overlay the mock plate first, followed by the plates infected with
the least amount of virus (1 O*), continuing in order to those plates infected with
the most amount of virus (1 03). Prepare enough media/agarose to overlay one to
two extra plates.
20. The plaques should be visible as holes in the monolayer of cells or l- to 2-mm
circular areas where the density of the monolayer appears lighter or heavier than
the surrounding area when the plates are held up to the light. The formation of a
plaque can be confirmed by circling the area with an ethanol soluble pen and
viewing under the microscope usmg the 10X objective. The plaque will appear
as a roughly circular area of dead cells. The plaques will be more difficult to
see than human-adenovirus plaques, and seeing them macroscopically is easier If
observed against a black background. We have a piece of black paper on the ceiling
near an overhead light and this seems to be crucial for seemg MAV- 1 plaques.
21. Wild-type vu-us generally grows to a titer of approx lo6 or lo7 PFU/mL; however, some mutants, depending on the nature of the defect, may grow to lo- to
lOO-fold lower titers than wild-type virus. The titer of the stocks may also
decrease upon multiple freeze-thaws, so we recommend aliquotting the large
stock of virus, especially if small amounts of vuus are needed to obtain the desired
multiplicity of infection (MOI).
References
1. Beard, C. W. and Spindler, K. R. (1996) Analysis of early region 3 mutants of
mouse adenovirus type 1 J Viral 70,5867-5874.
2. Smith, K., Y ing, B., Ball, A. O., Beard, C. W., and Spindler, K. R. (1996) Interaction of mouse adenovuus type 1 early region 1A protein with cellular proteins
pRb and ~107 Virology 224,184-197.
3. van der Veen, J. and Mes, A. (1973) Experimental infection with mouse adenovtrus in adult mice. Arch. Gesamte Virusforsch. 42,235-241.
4. Ishibashi, M. and Yasue, H. (1984) Adenoviruses of animals, in The Adenoviruses
(Ginsberg, H S., ed.), Plenum, New York, pp. 497-562.
5. Ball, A. O., Beard, C. W., Villegas, P., and Spindler, K. R. (1991) Early region 4
sequence and biological comparison of two isolates of mouse adenovirus type 1.
Virology 180,257-265
6. Kring, S. C., King, C. S., and Spindler, K. R. (1995) Susceptibility and signs
associated with mouse adenovnus type 1 infection of adult outbred Swiss mice. J
Virol. 69, 8084-8088.
7. Pirofski, L., Horwitz, M. S., Scharff, M. D., and Factor, S. M. (1991) Murine
adenovirus infection of SCID mice induces hepatic lesions that resemble human
Reye syndrome. Proc. Natl. Acad SCL USA 88,4358-4362.
102
9. Guida, J. D., Fejer, G., Pirofski, L.-A., Brosnan, C. F., and Horwitz, M. S. (1995)
Mouse adenovirus type 1 causes a fatal hemorrhagic encephalomyelitis in adult
C57BL/6 but not BALB/c mice. J. Viral. 69, 7674-768 1.
10. Ball, A. O., Williams, M. E., and Spindler, K. R. (1988) Identification of mouse
adenovirus type 1 early region 1: DNA sequence and a conserved transacttvatmg
function. J. Virol. 62,3947-3957.
11. Ball, A. O., Beard, C. W., Redick, S. D., and Spindler, K. R. (1989) Genome
organization of mouse adenovnus type 1 early region 1: a novel transcription map.
Virology 170,523-536.
12. Beard, C. W., Ball, A. O., Wooley, E. H., and Spindler, K. R (1990) Transcription mapping of mouse adenovnus type 1 early region 3 Virology 175,
8 l-90.
13. Cauthen, A. N. and Spindler, K. R. (1996) Sequence of the mouse adenovirus
type-l DNA encoding the lOO-kDa, 33-kDa and DNA binding proteins. Gene
168,183-187.
21. Song, B., Hu, S.-L., Darai, G., Spindler, K. R., and Young, C. S. H. (1996) Conservation of DNA sequence in the predicted major late promoter regions of selected mastadenoviruses. Virology 220,390-401.
22. Weber, J. M., Cai, F , Murali, R., and Burnett, R. M. (1994) Sequence and
structural analysts of murine adenovirus type 1 hexon. J, Gen. Vzrol. 75,
141-147.
22a.Meissner, J. D., Hirsh, G. N., LaRue, E. A., Fulcher, R. A., and Spindler, K.
R. (1997) Completion of the DNA sequence of mouse adenovirus type 1:
Sequence of E2B, Ll, and L2 (18-51 map untts). Virus Res. 51,53-64.
Construction of MA V- 1 Mutants
103
23. Wigand, R., Gelderblom, H., and Gzel, M. (1977) Biological and biophysical
characteristics of mouse adenovirus, strain FL. Arch. Vi&. 54, 13 1-142.
24. Hartley, J. W. and Rowe, W. P. (1960) A new mouse virus apparently related to
the adenovirus group. Virology 11,645-647.
25. Kapoor, Q. S. and Chinnadurai, G. (1981) Method for introducing site-specific
mutations into adenovirus 2 genome: construction of a small deletion mutant in
VA-RNA, gene. Proc. Natl. Acad. Sci. USA 78,2184-2188.
26. Stow, N. D. (1981) Cloning of a DNA fragment from the left-hand terminus of the
adenovirus type 2 genome and its use in site-directed mutagenesis. J. Viral. 37,
171-180.
27. Chinnadurai, G., Chinnadurai, S., and Brusca, J. (1979) Physical mapping of a
large-plaque mutation of adenovirus type 2. J. Virol. 32,623-628.
28. Larsen, S. H. (1982) Evolutionary variants of mouse adenovirus containing cellular DNA sequences, Virology 116,573-580.
29. Pettersson, U. and Sambrook, J. (1973) Amount of viral DNA in the genome of
cells transformed by adenovirus type 2. J, Mol. Biol. 73, 125-130.
30. Dunsworth-Browne, M., Schell, R. E., and Berk, A. J. (1980) Adenovirus terminal protein protects single stranded DNA from digestion by a cellular exonuclease.
Nucleic Acids Res. 8,543-554.
Generation of Adenovirus-Specific
Cytotoxic T Lymphocytes
Tim E. Sparer and Linda R. Gooding
1. Introduction
When using adenovirus asa vector for the delivery of transgenes,the induction of
a cytotoxic T lymphocytes(CTL) can limit the longevity of the transgeneexpression
(1,2). To detect the CTL responseto adenovirus proteins or inserted transgenesin a
mouse model, even with multiple in vivo immunizations, a secondary in vitro
restimulation is necessary.The CTL responseto adenovirus tends to focus on one or
a few immunodominant epitopes.Using secondaryin vitro restimulation of spleen
cells from primed mice as described here, an adenovirus-specific, MHC class
I-restricted CTL response has been generatedthat recognizes different immunodominant adenovirus antigens depending on the haplotype of the mice (34. Using
various adenovirus deletion mutantsor transfectantsexpressingindividual adenovirus proteins, it is possible to map the CTL antigens.With this knowledge, it is possibie to investigate methods for reducing or limiting the CTL response.
This method of generating adenovirus-specific CTL has also been employed to
investigate the immunoregulatory function of the adenovirus early region 3 (E3)
proteins. Adenovirus containsa cassetteof nine genesin E3, someof which function
to counteractboth the specific (i.e., CTL) and innate arms (i.e., tumor necrosisfactor)
of the immune response(5. Using this method of adenovirus-specific CTL generation, we have found that the E3 protein gpl9k can prevent the recognition of CTL
targets in some strains of mice but not in others, depending on the affinity of individual classI alleles for gpl9K (6). This can be an obstacle to mapping immune
responsesto adenoviruses that expressgpl9k. Using IFNy to overcome the gpl9k
effect allows CTL recognition of adenovirus infected targetsthat expressgpl9K (7).
In vivo gp19K hasnot been found by us to have an effect on the priming phase of the
CTL response,but the presenceof gpl9K can limit the development of Adenovimsspecific CTL when presentduring the secondaryin vitro stimulation (6).
From: Methods in Molecular Medicine, Vol. 21: Adenovirus
Methods and Protocols
Edited by: W. S. M. Wold Q Humana Press Inc., Totowa, NJ
106
2. Materials
1. Avertin (2% 2,2,2 tribromoethanol in H20, 0.5% tert amyl alcohol, filter sterilized) (Aldrich, Milwaukee, WI) for anesthetizing mice for intranasal inoculation.
2. CsCl-banded adenovirus (minimum of 10 PFU/mL).
3. Phosphate-buffered saline (PBS): 0.14 A4 NaCl, 1.5 mM KHZP04, 2.7 mM KCl,
8.1 mA4 Na2HP04, pH 7.35. This is used for diluting the adenovirus stocks and
for washing fibroblasts before adding trypsin for removal.
4. Dissecting instruments for harvesting spleens. This includes sterilized Petri dishes
with a loo-mesh stainless steel screen placed inside and a glass rod with flattened end
for pushing spleen cells through the screen to dissociate (Bellco, Vineland, NJ).
5. HEPES-buffered
Hanks balanced salt solution (HHR-5): 23.83 g HEPES
(Sigma, St. Louis, MO), 97.56 g Hanks balanced salts (Gibco-BRL, Gaithersburg, MD, into 10 L of H20) with 5% fetal calf serum (Hyclone). This is used as
a wash solution throughout this protocol.
6. Counting solution that allows for cell enumeration while lysing red cells: 0.01 g
crystal violet, 3 mL of glacial acetic acid. Mix these together first and adjust to
100 mL with PBS.
7. Complete RPMI: RPMI-1640
containing 10% fetal calf serum (Hyclone,
Logen, UT), 1% antibiotic/antimycotic
mixture (Gibco-BRL),
1 mA4
sodium pyruvate, 2 mM L-glutamine,
1% MEM nonessential amino acids
(Gibco-BRL),
5 x lop5 M 2-mercaptoethanol
(filter sterilized). The Lglutamine will last approx 1 mo in solution and the 2 ME will last only 2
wk in solution at 4C.
8. DME-IO: Dulbeccos modified Eagles medium containing 10% fetal calfserum,
2 mA4 L-glutamine. This medium is used to grow the SV40-transformed fibroblasts used as stimulator cells and as target cells.
9. DME-SF: Dulbeccos modified Eagles medium containing 2 mA4 L-glutamine
and 1% antibiotic/antimycotic.
10. cDME: DME containing 2 mM L-glutamine, 1% antibiotic/antimycotic
5 x 10e5A4
2-mercaptoethanol, 1% MEM nonessential amino acids (Gibco-BRL,), and 1 mM
sodium pyruvate.
11. Trypsin (0.25 mg/mL in PBS; Gibco-BRL): for removing the SV40-transformed
fibroblasts from Petri dishes.
12. 24-well Tissue-culture plates and 96-well V-bottom plates.
13. lOO- and 35-mm Petri dishes (Corning, Corning, NY).
14. Packard y-counter with additional statistical software.
3. Methods
3.1. Primary In Vivo Sensitization
(see Note 1)
1. For intranasal inoculation, three mice (6-8 wk old)/group are anesthetized with avertin
and inoculated with 25 & of CsCl-banded adenovims (10 PFU/mL) (see Note 2).
2. For intraperitoneal inoculation 2 x 10 PFU of virus is used per mouse. This dose
was shown to be the optimum dose to generate a strong adenovirus-specific
Adenovirus-Specific
Cytotoxic T Lymphocytes
107
CTL response (6) The presence or absence of the E3 gene product gp 19K m
the moculatmg virus has no effect on the quantity or spectftcity of CTL generated (3,6).
3.2. Maintenance
1 Most of the target cells used to analyze mouse CTL responses are SV40-transformed fibroblasts dertved from primary cultures of mouse embryo or adult kidney fibroblasts (8)
2. Grow SV40 tibroblasts to confluency m 100~mm plates at 37C and 8% CO,
Each loo-mm plate yields 2-5 x lo6 cells.
3 To remove cells, aspirate the medium and add approx 1 mL sterile PBS
4 Aspirate the PBS and add approx 1 mL trypsm (0.25 mg/mL m PBS).
5. After 2-3 min remove the cells and wash to remove the trypsm.
6 Subculture at least twice per week at a split ratio of I:20
3.3.2. Adenovirus
1. The day prior to splenocyte harvest, syngeneic (at least MHC identical, and
preferably from the same mouse strain as the spleen donor) trypsmlze SV40transformed fibroblasts and wash once in HHR-5 by centrifugation at 150g
for 10 mm
2. Place 1 x lo6 sttmulators m DME-10 onto 100 mm tissue culture dishes and
allow to adhere for at least 4 h (less time followmg trypsm treatment results m
decreased efficiency of vn-us mfectton)
3 Remove medium and replace with 4 mL DME-SF
4 Infect cells with 100 PFU/cell of human adenovirus. Allow the adenovnus to adsorb
for 2 h at 37C m 8% CO2 Remove the medium and replace with 6 mL of DME- 10.
5. After overnight mcubation at 37C and 8% CO,, harvest the stimulators with
trypsm and wash once with HHR-5.
108
109
One set of triplicate wells per target receives labeled target cells plus 50 pL of
cDME (medium only). These wells function as a measure of spontaneous release
of 5Cr from each target. Another triplicate of wells for each target will receive
lysis medium at the end of the incubation (maximal release). In total there are
eight sets of triplicate wells for each target (24 wells total: 3 wells maximal
release, 3 wells medium only, 3 wells 6O:l E:T ratio [stimulated], 3 wells
nonstimulated), 3 wells 2O:l E:T ratto (stimulated), 3 wells (nonstimulated), 3
wells 20: 1 (sttmulated), 3 wells (nonstimulated). Centrifuge the plate at 1508 for
2 min with low brake. Incubate the plates for 5 h at 37C at 8% CO,.
4. Notes
1 In order to generate CTL m vitro, it IS necessary first to prime the lymphocyte
population by inoculating mice with virus a mmimum of 8 d prior to harvest of
spleen cells for in vitro culture. Spleen cells from unprimed animals exposed to
adenovuus infected cells in vitro will not differentiate into CTL. This is why
SV40-transformed fibroblasts can be used as m vitro stimulator and target cells
without fear of generating a response to SV40 (9).
2 Intranasal maculations: Once the mice have been anesthettzed, the bolus of adenovnus is placed on the tip of the nose while holding the mouse up by the scruff of
skm under the chin. This IS important since it forces the mouth closed and prevents the majority of the moculum from going into the mouth. The concentration
of the adenovnus is crucial. We have found that even a log difference in titer can
prevent CTL priming. Also, we did not freeze-thaw these stocks in order to avoid
decreasing the titer.
3 Past experience indicates that the capacity to support m vitro generation of CTL
varies from one lot of fetal calf serum to another. Preliminary screening should
be done to identify a suitable lot that supports strong development of specific
CTL and not nonspecific cytolysis.
4. Secondary in vitro sensitization Nonstimulated cultures can either be incubated with
no stimulators or with uninfected stimulators Sirmlar results were achieved either way.
5. Care is required when working with radiolabeled chromium. This work should
be done using protective clothmg, gloves, and eye protection. Personnel also need
110
9.
to be monitored for exposure during this work. Addtttonally, any waste (solid or
liquid) generated should be disposed of m accordance with applicable instttutional guidelines
When using El A-deleted viruses to Infect target cells, mfectton should be with
1000 PFU/cell begmnmg 48 h prior to assay. Since these viruses lack the
transacttvatmg region of adenovitus, they require longer periods of time and
higher viral concentrattons to get complete vrral gene expression m mouse cells.
The protocol given here labels cells with chromtum overmght; this long a labelmg period is sometimes, but not always necessary (it will depend on the characteristtcs of the cells bemg used as targets). A mmtmum of 3 h 1srecommended
If the MHC class I haplotype of the target cells is among those that bmds to
gpl9K, then cells can be treated wtth 50 U of recombinant interferon-y (IFN-y)
(Genzyme, Cambridge MA) beginning immediately after vnus mfectton (7). IFNy treatment Increases levels of class I, overcoming the gpl9K blockade, but it
does not interfere with expression of viral protems. Although the degree of suppression of CTL recogmtion by gp 19K varies from one cell line to another, even
among cells of the same haplotype, m many SV40-transformed cells we have
observed a 50-60% drop m CTL recognition Alleles for which IFN-)I treatment
of target cells 1s advisable include Db, Ld, and Kd
Target cells. It 1scrucial that target cells not remain in air at room temperature for
too long as the spontaneous release of 51Cr increases the longer the cells are
exposed to temperature and CO2 fluctuattons
References
1. Yang, Y., Lr, Q., Ertl, H C., and Wilson, J. M. (1995) Cellular and humoral
mnnune responses to vtral antigens create barrters to lung-directed gene therapy
with recombinant adenovn-uses. J Viral 69, 2004-20 15
2. Yang, Y., Nunes, F A , Berencst, K., Furth, E E , Gonczol, E., and Wilson, J. M.
(1994) Cellular nnmuntty to vtral anttgens limits E 1-deleted adenoviruses for gene
therapy. Proc Nat1 Acad Scl USA 91,4407-441 I.
3. Rawle, F., Knowles, B. B., Ricciardi, R P , Brahmacheri, V , Duerksen-Hughes,
P., Wold, W. S M., and Goodmg, L. R. (1991) Specificity of the mouse cytotoxtc
T lymphocyte response to Adenovirus 5 Immunodommance
of E 1A depends on
H-2 haplotype. J Immunol. 146,3977-3984
4 Sparer, T., Wynn, S. G., Clark, D J , Kaplan, J. M., Cardoza, L. M., Wadsworth,
S. C , Smith, A E., and Gooding, L R. (1997) Generation of cytotoxtc T lymphocytes against immunorecessive epitopes after multiple nnmunizations with adenovirus vectors is dependent on haplotype. J Vlrol 71,2277-2284.
5 Wold, W S. M and Goodmg, L. R. (1991) Region E3 of adenovirus. a cassette of
genes involved m host unmunosurvetllance and virus-cell interactions Vzrology
184, l-8
111
7 Sparer, T., Trtpp, R. A, Ddlehay, D. L., Hermiston, T W., Weld, W S M., and
Gooding, L R. (1996) The role of adenovirus early region 3 proteins (19K, 10 4K,
14.5K, and 14.7K) m a murme pneumoma model J Viral. 70,243 l-2439
8. Goodmg, L. R. (1979) Specrfictties of killing by T lymphocytes generated against
syngeneic SV40 transformants. Studies employmg recombinants wlthm the H-2
complex. J. Immunol 122, 1002-1008.
9 Goodmg, L R (1977) Spectficittes of killmg by cytotoxtc T lymphocytes generated En VIVOand En wtro to syngeneic SV40 transformed cells. J Immunol 118,
920-927
9
Measurement of Macrophage-Mediated
of Adenovirus-Infected
Cells
Penelope J. Duerksen-Hughes
Cytolysis
1. Introduction
Macrophages are well-known for their ability to serve as phagocytes,
ingesting and destroymg microorgamsms such as bacteria, and for their function m antigen presentation. Less appreciated, perhaps, is the fact that macrophages are also capable of recognizing and destroying certain abnormal
self-cells; for example, some virus-infected and tumor cells are macrophage
targets. This chapter describes an assay procedure by which the macrophagemediated cytolysis of such cells can be measured.
In this procedure, macrophages are first activated with lipopolysaccharide
(LPS). Simultaneously, the target cells are prepared. This includes virus infection, if the targets are to be virus-Infected cells, and 5Cr labeling for all cells.
Then, the activated macrophages and labeled target cells are combmed at the
appropriate ratios m wells of 96-well plates and allowed to comcubate for an
appropriate period. At the end of this period, ahquots of media are removed
from each well and the amount of radioactivity released into this media is measured. This raw data is then used to calculate the percentage of lysed cells.
2. Materials
2.1. Effecfor
Cells
From
113
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114
Duerksen-Hughes
and Gooding
2.3. Assay
1 2 N HCl (room temperature).
2. Disposable borosihcate glass tubes
3 y-Counter.
3. Methods
3.1. Preparation of Effecfor Cells
3.1.1. Induce with Thioglycolla te
Four to five days before the experrment IS planned, inject between 1 and 5
male C3HeB/FeJ mice wtth 1S mL Brewers thioglycollate broth peritoneally,
depending on the size of the experiment planned. In general, one can obtain
between a little less than one and two 96-well plate(s) of macrophages per
mouse. The throglycollate broth induces the accumulatton of macrophages m
the perttoneal fluid, and provides the first step toward then actrvation.
Macrophage-Mediated
Cytolysls
1. Sacrifice the mice by CO, asphyxiation and lay them on their backs. Apply ethanol to the mldsectlon, then hft the skin with forceps and cut the skin vertically
and hortzontally such that the skin flaps can be opened to expose the abdomen.
2. Inject 10 mL of DMEM into each peritoneum using an 18 and l/2-gage needle.
Rock mice to allow the media to suspend the cells; one can also palpate the abdomen gently.
3. Insert a second needle and syringe mto a second site and withdraw the fluid Try
to avoid fat (which will clog the needle) and blood One should obtain between 5
and 10 mL of fluid per mouse
4 Put the fluid mto a 50-mL polypropylene centrifuge tube (macrophages may
adhere to other types of plastic, such as polystyrene) and centrifuge for 10 mm at
15Og m a bench-top centrifuge. Place tube on ice
5 Aspirate off the supernatant, flick the pellet (see Note 2), and add DMEM to the
tube The amount of DMEM added will depend on the estimated yield of macrophages per mouse (based on visual inspection of the pellet); approx 3 mL per
mouse IS a reasonable starting point Combine the tubes with macrophages (one
tube per mouse at this point) into a single tube.
6. Determine the concentration of macrophages usmg a hemocytometer Add media
to obtain a final concentration of 2 x 1O6macrophages/mL.
Duerksen-Hughes
116
and Gooding
out the media by gently touching the plasttc ttp to the mtersectron of the stde and
bottom of each well and allow the media to be suctroned out Repeat for each
well Add 100 p.L warm media to each well (a repeating pipettor or an 8-trp
ptpettor will make this step easier), rock the plates gently, then aspirate out the
media, and repeat three times
6 Add the LPS. Note that for each data point, it is usually necessary to measure
cytolysis m the presence both of macrophages activated with LPS and those not
exposed to LPS. Therefore, only half of your macrophages should be treated wtth
LPS. Aspirate out the last wash media from the wells and add 100 mL LPS (at
5 mg/mL) to each well to be treated; add 100 mL media to the other half of the
wells. If one has two plates of macrophages, tt 1susually easiest (and muumtzes
the chances of crosscontamination) to treat one plate with LPS and the other with
media. Also, add LPS to wells contammg those extra macrophages that will be
used for the supernatant control.
7 Incubate overnight m the CO, incubator.
3.2. Preparation
of Target Cells
The infection
is
Macrophage-Mediated
Cytolysis
117
5
6
7
8
For example, if you wanted to add 100 PFU/cell, and had a stock with lo9
PFU per mL, X would equal 100 (100 PFU/cell) and Y would equal lo9 ( IO9
PFU/mL m the vnus stock). One would therefore add 1 x 1O6x 100/l 0 = 0 100 mL
vu-us stock per plate
Somcate the vnus stocks for approx 1 mm to break apart clumps that frequently
develop and to create an even suspension of virus particles
In the biohazard tissue-culture hood, add virus to the plates usmg a Pipetman
Change pipet-tips between viruses
Gently swirl media and virus around m each of the 60-mm plates to ensure an
even distribution of the virus.
Return the 60-mm plates to the incubator (37C, 8% COZ) for 90 mm (see
Note 5).
9 In the biohazard hood, aspirate the vu-us and medium from the cells and rinse the
cells twice with 2 mL serum-free DMEM (prewarmed to 37C) (see Note 6)
10 Add 2 mL DMEM (+lO% FCS) to each of the plates and return the plates to the
incubator. Allow the mfectron to proceed for approx 16 h; the actual time can be
adlusted as needed. Too long an mcubatron time can result in cytotoxicity,
whereas too short a time can result m insufficient expression of vuus proteins.
Human cells will require less time than will rodent cells
118
Duerksen-Hughes
and Gooding
2. Centrifuge cell suspensions at 15Og for 10 mm. Decant the supernatant, flrck the
pellet, and add approx 2 mL media.
3 Determme the cell concentratron usmg a hemocytometer
Add the reqmred
amount of medra to bring the final concentration to 2 x lo5 cells/ml
Add 100 pL of the radiolabeled target cells to each well with macrophages.
A repeating ptpettor ~111make thrs step easier. It ISalso recommended that one
add the targets to the unactivated macrophages first, then to the activated macrophages. This minimrzes the chance of accrdentally transferrmg LPS to the
unacttvated macrophages.
3.3.2 Prepare Controls
Four types of controls are necessary; the first three may be best prepared
using a new 96-well plate.
1 The maxtmum control determrnes the maximum radroactrvrty that can be released
by an aliquot of labeled targets. At this point, to each of three wells of a new
plate, add 100 pl.. target cells This should be done for each of the target cell
populations to be tested.
2. The buffer control determines the amount of radtoacttvity that ~111 leak out m
the absence of lysts. At thts point, add 100 p.L target cells plus 100 p.L medra
to each of three wells This should be done for each of the target-cell populations to be tested.
3 The supernatant control is helpful m drscrimmatmg between ktll mediated by
macrophage contact and kill mediated by soluble mediators released by macrophages. Harvest and combme the conditioned media from the wells contaimng
the extra activated macrophages. Store this condtttoned media on ice Add 100 pL
of the target cells plus 100 pL of the conditioned media to each of three wells
Thts should be done for each of the target-cell populations to be tested
4 The acttvatton control helps to determme that any effects seen are m fact dependent on macrophage activation Because you have already set up separate plates
with activated and unacttvated macrophages, this control has already been taken
care of.
Macrophage-Mediated Cytolysis
119
3. Centrifuge microtiter plates at 150g for 10 min. Spin the samples with a low
brake to keep the cell pellets intact.
4. Transfer 100 pI, supernatant from each well into separate borosihcate glass tubes
using a ~200 Pipetman. The same pipet tip can be used for each set of triplicates
within the row, but change tips between these sets. Do not release a sample of
supernatant back into a well to try a transfer again; also, do not touch the bottom
of the plate with a pipet ttp. Etther of these actions may pick up radtoacttve,
unlysed cells and give a falsely high reading.
5. Determine the radtoacttvity of these cellular supernatants on a y-counter.
6 To analyze the percent specific lysis induced by the macrophages, average the
trtphcate values and use the following equatton (see Note 9). [(experimental spontaneous release)/(maxtmum - spontaneous release)] x 100. The spontaneous
release is that found in the buffer control wells, the maximum release IS that
found in the maximum control (or HCI-treated) wells.
7 Graph and analyze your data For examples of this technique, see refs. 2-4.
4. Notes
1 The part of this assay that 1sprobably the most drfficult to reproducibly replicate
is the state of the macrophages. We have found that even trace amounts of endotoxin present in the media used during the preparatory steps (Subheading 3.1.2.)
can cause the expertment to fail. Therefore, we recommend purchasing serum
from lots with an absolute mimmum of endotoxin, and to use media purchased m
liquid form from the supplier in order to mmimtze the possibility of laboratoryinduced contaminatton with endotoxin
2. Resuspension of the pellet at this point is a fairly crttical step, as macrophages
adhere to each other easily and once clumps form they are difficult to dissoctate.
We have found that the best way to accomplish this is to vigorously flick the
pellet (and, If necessary, even bang the centrifuge tube against the countertop) to
loosen the cells before addmg the media; once media ts added, any clumps present
are probably there to stay.
3. The high level of protein m DMEM + 10% FCS inhibtts attachment of adenovrrus to Its cellular receptor Hence, It is critical to use serum-free medra or media
with a low percentage of serum during the mfection.
4 The PFU/cell IS dependent on the cell type In general, a high multlpllclty of
infection IS needed for complete infection of mouse cells and rat cells (100-200
PFU/cell). Human cells require a much lower multiplicity of Infection (S-20 PFU/
cell) for complete infection.
5 One and a half hours is generally adequate for mfection However, tf the cell lme
you are working wtth is difficult to infect, you can increase the amount of incubation
time with the vn-us to 2 h. It is inadvlsable to incubate cells m serum-free DMEM
for longer than 2 h, as the cells will begin to lyse because of serum starvatton.
6. To ensure vu-us inactivation after removal from the plate, one may mclude in the
trap an agent such as Wescodyne Also, solid waste should be collected as biohazard waste and autoclaved before disposal,
120
Duerksen-Hughes
and Gooding
7. The protocol grven here labels cells with chrommm overnight; this long of a labelmg penod 1ssomettmes, but not always, necessary (tt will depend on the characteristics of the cells being used as targets). A mmimum of 3 h is recommended.
8. Care 1s required when workmg with radiolabeled chrommm. This work should
be done using protective clothing, gloves, and eye protection. Personnel also need
to be monitored for exposure durmg thts work Addtttonally, any waste (sohd or
liquid) generated should be drsposed of m accordance wtth apphcable institutional gutdelmes.
9 The spontaneous release should be no more than 30% of the maximum release. A
high spontaneous-release value could indicate that the targets are poorly viable,
or that you have allowed the assay to run too long
References
1 Whitmore, A C and Goodmg, L. R. (198 I) Spectfictttes of ktllmg by T lymphocytes generated against syngeneic SV40 transformants: cross-reacttvtty of the
H-2Kbm1 through Kbm4H-2 mutant alleles in Kb-restricted SV40-specific ktllmg
J Immunol 127, 1207-1211.
2. Duerksen-Hughes, P. J , Day, D. B., Laster, S M , Zachartades, N. A., Aqumo,
L , and Goodmg, L. R. (1992) Both tumor necrosts factor and mtrtc oxide parttctpate in lysis of Simian Virus 40-transformed cells by activated macrophages
J Immunol. 149,2114-2122.
3. Laster, S. M., Wood, J. G., and Gooding, L. R. (1988) Target-mduced changes in
macrophage migration may explam differences in lyttc sensttivtty among stmian
virus 40-transformed fibroblasts. J Zmmunol 141,221-227.
4. Laster, S. M and Goodmg, L. R. (1990) Evidence that a target-derived soluble
factor IS necessary for the selecttve lysts of SV40-transformed fibroblasts by acttvated mouse macrophages. J. Immunol 144, 1438-1443
1. Introduction
To mmimtze or prevent the spread of an acute vnus mfectton, the anttvrral
immune response must detect and lyse virus-mfected cells before virus rephcates or 1s released from the host cell. The immune response has developed
both Innate and specific responses to meet this objective. Virus-Infected cells
are lysed by cells of the innate immune response such as acttvated macrophages and natural killer cells, and by cytokines like tumor necrosis factor (TNF)
(1,2). Here, we will discuss methods we developed to measure the cytotoxic
capacities of TNF against adenovnus-infected cells.
The TNF cytotoxtcrty assay our laboratory developed requires 3 d from
beginning to end. On day one, cells are mfected with adenovnus and labeled
with radioactive sodium chromate (NaZ5Cr04). TNF assayplates are prepared
and infected cells are added to TNF on d 2. On d 3, the assay IS harvested,
counted on the y-counter and the percent specific lysts induced by TNF is
determined. Because not all cells are readily infected with human adenoviruses
(especially mouse cells), a control to determine the extent of mfectton is
included m every experiment.
2. Materials
1 Adenovirus
stocks: Mamtam
ahquots of viruses that will be used repeatedly over a short period of time at
-20C Btosafety level 2 precautrons, including gloves and protective clothing,
should be observed whtle workmg with adenovtrus or adenovtrus-infected cells,
2. Human recombmant TNF (Genzyme, Frammgham, MA) (5400 U/pL) Store
at -7OC m ahquots (ZO-pL) to mmtmrze repeated freezing and thawing
From
721
722
3 NaZ5C!r0, (1000 Cl/g) (DuPont NEN, Boston, MA). Store at 4C This compound
emlts y-radlatlon, thus appropriate lead shielding and protective gloves and clothmg should be employed for mmlmal exposure and protection to personnel
4 NIH-3T3 mouse fibroblast cells (ATCC, Rockvllle, MD) Mamtam m D-MEM
(see below) at 37C, 8% CO,
5 Culture media for mouse cells consists of DMEM (Dulbeccos modified Eagles
medmm), containing 5% glutamine, supplemented with 10% fetal calf serum
(FCS). Infection of mouse cells requires DMEM with 5% glutamme only
(referred to as serum-free DMEM)
6 Sterile flat-bottomed 96-well plates (see Note 1)
7 Trypsm, stored at -20C.
8 Refrigerated centrifuge, with swinging buckets and mIcrotIter plate carriers
9 Sterile 60-mm plastic tissue-culture plates.
10 Sterile and nonsterile phosphate-buffered salme (PBS), pH 7 4, at room temperature.
11. HBSS (Hanks balanced salt solution) or other suitable wash solution (4C)
12. Disposable borosillcate glass tubes
13. 15-mL Centrifuge tubes
14 2 N HCI (room temperature)
15. y-Counter
16. Hemocytometer
17 g-Well chamber slides (glass or plastic) suitable for tissue culture
18. M73 anti-El A antibody (ATCC)
I9 FITC-conjugated antlmouse antibody
3. Methods
as a smaller volume of media ensures adherence. Allow cells to adhere (at least
h) (see Note 2).
of d 7
1 Remove virus aliquots from freezer (-20C). Thaw viruses quickly at room
temperature, then immediately put thawed ahquots on Ice. Before infecting
cells, somcate virus allquots for 30 s Somcatlon dlssoclates virus particles
from cellular debris created during virus propagation, thus increasing the
efficiency of infection.
2 In a tissue-culture hood reserved for biohazard use, remove DMEM media from
the cells and briefly rinse each of the plates two times (5 s each time) with 3 mL
of serum free DMEM (prewarmed to 37C) (see Note 3).
123
Cells
3 Add 1.5-2 mL serum free DMEM to each 60-mm plate. Rotate the plates, covermg the entire bottom of the plate with serum-free DMEM.
4 Given that the titer of the virus stock 1s known, determine the volume of vrrus
needed for 100 PFU/cell using the equation below (see Note 4) For example, if
the virus titer is I O9PFU/mL (plaque-forming units/ml), and there are 1O6NIH3T3 cells to be infected at an MO1 (multiplicity of infection) of 100 PFU/cell,
then 0.100 mL of virus would be added to cells
([(l x lo6 cells) (XPFWcell)] I(YPFUImL)}
{[(l x lo6 cells)
(100 PFU/cell)] / ( lo9 PFU/mL)} = 0.100 mL vu-us to add to cells
6
7.
8.
9.
10
of d 2
124
sample
sample
sample
sample
1 2 3 4 5 6 7 8 9101112
M-mum
Spontaneous
0 002 U TNF/ml
0 02 U TNF/ml
0 2 U TNF/ml
2 U TNF/ml
A
E?
C
D
E
F
50 M, 50 C (100 HCl)
150 M, 50 C
100 M, 50 TNF
( 0 002 U/ml),
50 C
100 M, 50 TNF
( 0 02 U/ml),
100 M, 50 TNF
( 0 2 U/ml),
100 M, 50 TNF
( 2 U/ ml), 50 C
-b
50 C
50 C
_____)
______)
,-b
20 U TNF/ml
100 M, 50 TNF
( 20 U/ ml), 50 C -+
200 U TNF/ml
100 M, 50 TNF
( 200 U/ml),
50 C .-W
Fig. 1 Layout of mtcrottter plate for TNF assay. Numbers mstde the large
rectangle indicate the amount in mrcrolrters for each well M, medta; C, mfected
cells; HCl, hydrochlortc acid (added on d 3). Row A is maxtmum release, Row B
IS spontaneous release, and Rows C-H are expertmental values, with mcreasmg
concentrations of TNF. Each microttter plate can be used for up to four dtfferent
viruses
rus-Infected cells. Usmg an s-tip ptpetman, fill the plate with DMEM as follows
(Fig. 1).
Row A. 50 p.L medra
Row B: 150 pL media
Rows C-H* 100 $ medra
2 Add hrTNF as follows (Fig. 1).
Row A: none
Row B: none.
Row C: 50 p.L of the 0.002 U/mL concentratron of hr TNF to each well.
Row D: 50 pL of the 0.02 U/mL concentratton of hr TNF to each well.
Row E: 50 p.L of the 0.2 U/mL concentratron of hr TNF to each well.
Row F: 50 pL of the 2 U/mL concentratton of hr TNF to each well.
Row G: 50 pL of the 20 U/mL concentratron of hr TNF to each well
Row H: 50 pL of the 200 U/mL concentratton of hr TNF to each well.
3. At this stage, all wells should contam 150 PL of TNF and/or medra, wrth
the exception of Row A, which will contain 50 nL of media only. 100 pL
of 2 N HCI will be added to Row A on d 3 to give maximum release values. Row B, which contains no TNF, gives values for spontaneous release
(Fig. 1). Store the microtiter plates at 4C unttl vrrus-Infected cells are
added to them.
Cells
125
11 Add 50-100 pL of each virus sample to two chambers on the chamber slide
These samples will be used to determine the percentage of cells that was infected
by that particular adenovtrus construct (see Subheading 3.4.). Incubate the
infected cells m the chamber slides overnight at 37C at 8% CO*
1 From this point, the experiment can be carried out in a nonsterile environment
such as a lab bench. Add 100 pL 2 N HCI to each of the wells in Row A. Mix the
contents of the well thoroughly, by pipetting up and down in each well, to ensure
complete lysis of cells Change pipet tips in between each well.
2. Centrifuge mtcrotiter plates at 250g for 10 min, usmg a low brake to keep the
cell pellet intact.
3. Transfer 100 pL of supernatant from each well into separate borosilicate glass
tubes. Do not disrupt the cell pellet. The same pipet ttp can be used for each of
triplicates within the row, but change tips between Row A and Row B, Row B
and Row C, and so on (see Note 7).
4 Count the cellular supernatant on a y-counter.
5. To analyze the percent specific lysis induced by TNF, average the triplicate
values for each row of each experimental, and use the following equation
(see Note 8).
726
3.4. Determining
Percent
infection
(* SE,,,,*) + ti
of Cells-d
1 While the microtiter plates are being centrifuged, examme the infected cells m
the chamber slides underneath a microscope. Observe and determine the percentage of cells that are adherent. Infected cells show a rounded morphology, whereas
uninfected cells are adherent
2. Remove media from each chamber and then remove the wells of the chamber
slide. Rinse the cells twice m a Coplm jar filled with dPBS Remove excess dPBS
3 Incubate the slide m absolute cold methanol (-2OC) for 10 min At this point, the slides
can be stored at -2OC for up to 1 wk without harmmg the sample. The shdes should be
stored in a new tissue-culture plate after it has been sealed with parafllm to prevent drymg
4. Rehydrate the sample m room temperature PBS for 5 mm
5 In Subheading 3.2.2., each sample of vnus-infected cells was plated mto two
wells on a chamber slide. For each vnus, add an anti-adenovnus antibody to one
well (our laboratory has used the M73 anti-ElA antibody [ATCC) for the primary antibody), and PBS to the other well. The cells incubated with PBS are a
negative control, measuring nonspecific bmding of the secondary antibody.
6 Rinse spot plates three times in room temperature PBS for 5-min mtervals
7 Remove excess PBS, drying around the sample. Add a FITC-conjugated rabbit
antimouse antibody to each sample and Incubate samples for 30-60 mm at 4C
8. Rmse three times m PBS and remove excess PBS
9. Put a few drops of glycerme on the glass slide, then put a cover slip on, pressmg
out any bubbles. Seal with nail pohsh
10 Examine cells under a fluorescent microscope to determine the percentage of mfection.
4. Notes
1. Modifications for suspension cells
a. Day 1: Use an appropnate volume of suspended cells for 1 x 1O6cells into 10 mL
serum-free DMEM m a 15-mL conical tube Centrifuge at 250g for 10 mm,
aspirate off medium, and resuspend cell pellet m serum-free DMEM Add
virus to the cells, and incubate at 37C for 1 h, resuspendmg cells gently
every 15 mm. Centrifuge the tube again for 10 mm at 25Og. Decant the serumfree DMEM and resuspend suspenston cells m 4 mL regular DMEM, and
transfer infected cells to a 25-cm3 flask. Add 200 pCi Na251Cr0, to each flask
and incubate overnight at 37C m 8% CO2
4.
7.
8.
9.
10
Cells
128
References
1 Goodmg, L R., Elmore, L W , Tollefson, A E , Brady, H A., and Wold, W S
M. (1988) A 14,700 MW protein from the E3 region of adenovuus mhibits
cytolysts by tumor necrosis factor Cell 53,34 l-346
2 Goodmg, L R , Ranheim, T. S., Tollefson, A E , Aqumo, L , Duerksen-Hughes,
P , Horton, T. M , and Weld, W. S M. (1991) The 10,400- and 14,500-dalton
proteins encoded by region E3 of adenovirus function together to protect many
but not all mouse cell lines agamst lysis by tumor necrosis factor J Vwol 65,
4114-4123
11
Role of Natural Killer (NK) Cells
in Immunity to Adenoviruses
John M. Routes
1. Introduction
Natural killer (NK) cells have become increasingly recognized as integral
members of the host cellular immune response. The purpose of this section
is to review the general biology of NK cells, their role in anttadenoviral
immunity, and outline the most commonly used method to assessNK-cell
lytic function.
729
and Protocols
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130
Routes
131
The ElA gene solely governs the difference in the cytolytic susceptibility
between Ad12- and Ad2/5transformed cells. In contrast to Ad12 El A, expression of Ad2/5 El A gene products in either Ad-infected or Ad-transformed
rodent cells induces an increased sensitivity to NK-cell lysis (28-30). Interestingly, although Ad2/5transformed human cells are sensitive to NK-cell lysis,
AdY54nfected cells are not (31,32). This inability of ElA produced in the
context of Ad-infection to induce susceptibility to NK-cell lysis might contribute to their proclivity to cause persistent infections in humans.
The capacity of Ad2/5 ElA to induce susceptibility to NK-cell lysis is relevant to the low oncogenicity of Ad2/5-transformed cells or Ad2/5 ElAexpressing tumor cells in vivo (33-38). For example, in vivo depletion of rat
NK cells with antiasialo GM1 antibody enhances the tumorigenicity of Ad2transformed rat cells without affecting T cell proliferative responses (39).
Transfection of the Ad5 El A gene into highly oncogenic sarcoma cells
increases their susceptibility to NK-cell lysis with a concomitant reduction in
their tumorigenicity. In vivo depletion studies with antiasialo GM1 show that
rejection of these El A-expressing tumor cells is NK cell dependent (36,38).
Other studies show that the inability of newborn rats to reject Ad5 ElAexpressing sarcoma cells is related to a developmental lack in NK-cell lytic
function. Maturation of NK lytic function with time correlates with the ability
to reject El A-expressing sarcoma cells in vivo (37). In summary, these data
suggest that the difference in oncogenicity between Ad12 and Ad2/5 may be
explained at least in part by the capacity of NK cells to reject Ad2/5- but not
Ad124ransformed cells in vivo. NK-cell lytic activity appears important in
mediating the rejection of Ad2/5 El A-expressing tumor cells.
7.3. Molecular Basis for NK-Cell Killing
of Ad215 ElA-Expressing
Cells
There are many possible explanations for the induction of cytolytic susceptibility by Ad2/5 El A and lack thereof by Ad12 El A. The difference in the
susceptibility between Ad1 2- and Ad2/5-transformed cells to lysis by NK cells
could be the result of a lack of NK cell binding and target-cell recognition of
Ad12-transformed cells or enhanced binding of Ad2M-transformed cells. However, early studies indicated that both Ad2- and AdlZtransformed cells are
equivalently bound by NK cells (25). An alternative hypothesis, which is supported by a recent study, suggests that Ad2/5 ElA sensitizes target cells to
destruction by killer cells independently of cell-surface recognition phenomena.
Cytotoxic lymphocytes, including NK cells, kill target cells through two
major mechanisms. One pathway involves degranulation and secretion of the
pore-forming protein, perforin. Alternatively, there is a nonsecretory pathway
that relies on inducible expression of fas ligand on killer cells interacting with
132
Routes
Immunity
733
cytolytic resistance by IFN rests with its capacity to increase the surface
expression of class I MHC Ags on target cells (58). However, again this relationship does appear to apply m the Ad-system.
IL-2-activated rat LAK kill Ad12-transformed rodent cells. IFN-treatment
of these cells increases class I MHC without changing the sensitivity of these
cells to lysis by LAK (39). IFN-activated NK cells nonselectively kill Ad2/5infected and uninfected human cells. IFN-treatment of uninfected target cells
induces class I Ag expression and reduces their susceptibility to NK-cell lysis.
In contrast, although IFN-treatment of Ad-infected cells increases class I Ag
expression to a similar extent as IFN-treated, uninfected cells, it does not protect infected cells from NK-cell lysis (59). The capacity of Ad to block the
protective effect of IFN is solely dependent on the expressron of El A gene
products, does not involve ElA peptidtilass I binding, and maps to the ~300
binding site of E 1A (60,61).
1.4. Measurement
Routes
134
2. Materials
2.1. Materials for Isolation
of Rodent Mononuclear
Cells
1. RPMI-10% FCS: RPM1 media (Gibco/BRL, Gaithersburg, MD, cat. no. 320/
1870AJ), 10% fetal calf serum (FCS), heat inactivated at 56C for 1 h, 2 mM
L-glutamine, pemcillin G (1000 U/mL) and streptomycin (100 pg/mL).
2. RBC lysing buffer: NH&I (8 29 g/L), KHC03 (1 .O g/L), and EDTA (0 0372 g/L).
Add to 1000 mL, filter with 0.22~pm filter, aliquot (85 mL) m bottles and refrigerate. Add 15 mL endotoxin free, heat inactivated FCS prior to use.
3. Hanks balanced buffer solution (HBBS).
4. Sterile scissors and forceps.
5. loo-Mesh wire sieve.
6. Freshly removed spleens from mice, hamsters, or rats (2-5-mo old).
7. Equipment and reagents for counting viable cells (e.g., trypan blue 0.1%
and hemocytometer).
RPM&IO, HBBS.
Ficoll-Hypaque solution (Ficoll-Paque, Pharmacia, Piscataway, NJ).
Heparin sulfate (1000 U/mL)
Equipment for drawing blood. Caution: Standard biosafety procedures must
be followed when using human blood. Donors should screened by serology
for hepatitis B, hepatitis C, and HIV-l prior to isolation of blood mononuclear cells.
2.3. Materials
of Mononuclear
735
1. Remove spleen under sterile conditions and place into a 60 x 15mm Petri dish
containing 5-10 mL of cold HBSS.
2. Cut spleen into half, and puncture repeatedly with 19-gage needle. Remove
splenocytes by repeatedly perfusing spleen with cold HBSS solution using 6-mL
syringe and 1g-gage needle. Remainder of splenocytes may be removed by teasing and crushing with forceps. Process is complete when only predominately
white, fibrous trssue remains.
3. Centrifuge (200g for 10 min). Resuspend pellet in 5 mL cold RBC lysing solutton on ice for 5 min. Add 10 mL HBSS, and centrifuge (200g for 10 min). Wash
pellet twice with HBBS.
4. Resuspend pellet with 5-l 0 mL RPMI- 10. Pass remaining cells through 100-mm
mesh wire sieve (2OOmm nylon mesh may be substituted), and count viable cells.
Resuspend splenocytes at the following concentrations: mouse: 4 x 10 cells/ml;
hamster or rat: 2 x lo7 cells/ml (see Note 1).
3.2. Isolation
of Mononuclear
from Human-Peripheral
Cells
Blood
3.3. Target-Cell
Preparation
1. l-2 x 1O6target cells are passaged into fresh media the night before the cytolysis
assay (see Note 3).
Routes
136
2. Prepare approx l-3 x lo6 target cells into a single cell suspension in RPMI-10.
3. Centritbge at 2008 for 10 min, pour off supernatant, and gently resuspend cells
(do not vortex) in remaining 100-200 $ of media. Add 100 $ of heat-mactivated, LPS-free FBS.
4. Add 100 pCi 51Cr, gently resuspend, and incubate in 37C water bath for 1 h.
Gently resuspend every 15 min. Increased incubation times (1.5 h) and/or 5Cr
(up to 200 PC) may be added if cells insufficiently label (see Note 6).
5. 5Cr-labeled target cells are washed two times in HBSS (15 mL). Resuspend pellet in 10 mL of RPMI- 10 and incubate additional 30-45 min (mix every 15 min)
in 37C water bath. (This additional purging step decreases the spontaneous
release of radiolabel) (see Note 5).
6. Centrifuge, resuspend pellet in complete media; count viable cells by trypan blue
exclusion, and resuspend cells at a final concentration of 5 x 1O4cells/ml. For
most targets, IO targets/well are used in cytolysis assays.
137
4. Notes
1. The following are average yields of mononuclear cells: human peripheral blood,
1 x IO6 mononuclear cells/ml blood; hamster, 4-6 x lo7 mononuclear cells/
spleen; immunocompetent mouse, 8-10 x lo7 mononuclear cells/spleen; nude
mouse, 5 x lo7 mononuclear cells/spleen; immunocompetent rat, 1 x lo8 mononuclear cells/spleen; nude rat, 4 x lo8 mononuclear cells/spleen.
2. Percent specific release (target-cell killing) of NK-susceptible cell lines (YAC-1
or K562) is usually 30% or higher at the highest E:T ratio. However, NK
activity from human-blood-mononuclear
cells is donor variable. Common
reasons for consistently low specific release of susceptible targets include
not promptly removing mononuclear cells from Ficoll-Paque, extended incubation times in RBC lysing solution, incubation of microtiter plates at room
temperature, and incorrect counts. Under certain circumstances, assay time may
need to be extended.
3 NK cells present in murine spleens have much lower resting NK-cell lytic activity than hamster or rat. Therefore, it is usually necessary to activate the NK cells
in vivo by administermg 100 pg of poly-IC (Sigma, St. Louis, MO) ip the night
prior to the isolatton of mononuclear cells. (Store m 1-mL ahquots at 100 pg/mL
in HBSS in -20C freezer )
4. The total releasable counts calculated from each cell line should be at least 1000 cpm
above background. If fewer counts are obtained, increase the amount of label or
labeling time. Alternatrvely, one can increase the number of target cells added
per well.
5. Percent spontaneous release values should be noted for each target-cell lure. Values above 30% are unacceptable. The purge step should eliminate high-spontaneous-release values.
6. The inability of a target-cell line to properly label or high-spontaneous-release
values may indicate improper cell-culture conditions prior to the assay or mtcrobial contamination. Particular attention to maintaining healthy cell cultures is
critical to the success of the assay. Cell lines should be routinely screened for
contamination with Mycoplasma.
Acknowledgments
I want to thank Larry Borish and James Cook for critical
manuscript and Mary Peterson for secretarial assistance.
review of this
References
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an
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Routes
138
4. Koneru, B., Jaffe, R., Esquivel, C. O., Kunz, R., Todo, S., Iwatsukr, S., and Starzl,
T. E. (1987) Adenoviral infections in pediatric liver transplant reciprents. J Am
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6) human adenovirus. Proc. Natl. Acad. Sci. USA 76,6606-6610
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8 Trentin, J. J., Yabe, Y., and Taylor, G. (1962) Quest for human cancer viruses
Science 137,835-84 1.
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human adenovirus types 12 and 18. Proc. Natl. Acad. SCL USA 48,2051-2058
10. Allison, A. C., Berman, L. D., and Levey, R. H. (1967) Increased tumor induction
by adenovirus type 12 in thymectomized mice and mice treated with anttlymphocyte serum. Nature 215, 185-187.
11. Gallimore, P. H. (1972) Tumor production in mnnunosuppressed rats with cells
transformed in wtro by adenovirus type 2. J. Gen Virol. 16,835-840.
12. Cook, J., Lewis, A., Jr., and Kirkpatrick, C. (1979) Age-related and thymusdependent rejection of adenovirus 2-transformed cell tumors in the Syrian hamster. Cancer Res. 39,3335-3340.
13. OShea, J. and Ortaldo, J. R. (1992) The biology of natural killer cells. insights
into the molecular basis of function, m The Natural Killer Cells (Lewis, C. E. and
McGee, J. O., eds), Oxford University Press, Oxford, pp. 2-27.
14. Philips, J. and Lanier, L. (1986) Dissection of the lymphocyte-activated killer
phenomenom: relative contribution of peripheral blood natnral killer cells and T
lymphocytes to cytolysis. J. Exp. Med. 164, 2133-2141.
15. Trinchieri, G., Matsumoto-Kobayashi,
M., Clark, S. C., Seehra, J., London, L.,
and Perussia, B. (1984) Response of restmg human peripheral blood natural killer
cells to interleukm 2. J. Exp. Med 160, 1147-l 169.
16. Ellis, T., McKenzie, R., Smuns, P., Helfrich, B., and Fisher, R. (1989) Induction
of human lymphokine-activated killer cells by IFN-a and IFN-y. .I Zmmunol. 143,
4282-4286.
17. Dunn, P. L. and North, R. J. (199 1) Early gamma mterferon production by natural
killer cells is important in defense against murme listeriosis. Infect Zmmunol. 59,
2892-2900.
19. Orange, J. S., Wang, B., Terhost, C., and Biron, C. (1995) Requirement for natural killer cell-produced interferon y in defense against murine cytomegalovuus
139
infection and enhancement of this defense pathway by interleukin 12 administratron. J, Exp. Med. 182, 1045-1056.
20. Scharton, T. M. and Scott, P (1993) Natural killer cells are a source of IFN-y that
drives differentiation of CD4+ T cell subsets and induces early resistance to Leishmania major in mice. J. Exp. Med 178,567-577.
21 Stitz, L., Baenziger, J., Pircher, H., Hengartner, H., and Zmkernagel, R. M.
(1986) Effect of rabbit antiasialo GM1 treatment in vivo or with anttasialo
GM1 plus complement in vitro on cytotoxic T cell activities. J. Zmmunol. 136,
4674-4680.
22 Kos, F. J. and Engleman, E G. (1995) Requnement for natural hller cells m the
induction of cytotoxic T cells. J Immunol 155, 578-584.
23. Biron, C. A., Byron, K S., and Sullivan, J. L. (1989) Severe herpes virus infections in an adolescent without natural killer cells. N Engl. J Med. 320, 173 1.
24. Welsh, R. M. (1986) Regulation of virus infections by natural killer cells. Nat.
Immun. Cell Growth Regul. 5, 169-199.
25. Raska, K., Jr. and Gallimore, P. H. (1982) An inverse relation of the oncogenic
potential of adenovirus-transformed cells and their sensitivity to killing by syngeneic natural killer cells Virology 123,8-18.
26. Cook, J. L., Hibbs, J. R., Jr., and Lewis, A. M., Jr. (1982) DNA virus-transformed
hamster cell-host effector cell interactions: level of resistance to cytolysis correlated with tumorigemcity. Int. J Cancer 30,795-803.
27. Soddu, S. and Lewis, A. M., Jr. (1992) Driving adenovirus type 12-transformed
BALB/c mouse cells to express high levels of class I major histocompatibility
complex proteins enhances, rather than abrogates, their tumorigemctty. J. Viral.
66,2875-2884.
28. Sawada, Y., Fohring, B , Shenk, T E., and Raska, K., Jr. (1985) Tumorigenicity
29.
30.
3 1.
32
33.
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Routes
34. Lewis, A. M., Jr. and Cook, J. L. (1985) A new role for DNA virus early proteins
in viral carcinogenesis. Science 227, 15-20.
3 5. Kenyon, D. J. and Raska, K., Jr. (1986) Region El A of highly oncogenic adenovnus 12
in transformed cells protects against NK but not LAK cytolysis. viro/ogv 155,644654.
36. Walker, T. A., Wilson, B. A., Lewis, A. M., Jr., and Cook, J. L. (1991) E IA
oncogene induction of cytolytic susceptibility eliminates sarcoma cell tumorigenicity. Proc. Natl. Acad Sci. USA 88,6491-6495.
37. Cook, J. L , Ikle, D. N., and Routes, B. A. (1995) Natural killer cell ontogeny in
the athymic rat. Relationship between functional maturation and acquired resistance
to ElA oncogene-expressing sarcoma cells. J. Immunol. 155,5512-5518.
38. Cook, J. L., Wilson, B. A., Wolf, L. A., and Walker, T. A. (1993) El A oncogene
expression level in sarcoma cells: an independent determinant of cytolytic susceptibility and tumor rejection. Oncogene 8,625-635.
39. Kenyon, D. J., Dougherty, J., and Raska, K., Jr. (1991) Tumorigenicity of adenovirus-transformed cells and their sensitivity to tumor necrosis factor a and NW
LAK cell cytolysis. Vzrulugy 180,8 18-821.
40. Arase, H., Arase, N., and Saito, T. (1995) Fas-mediated cytotoxicity by freshly
isolated natural killer cells. J Exp. Med. 181, 1235-1238.
41. Vujanovic, N. L., Nagashima, S., Herberman, R. B., and Whitestde, T. L. (1996)
Nonsecretory apoptotic killing by human NK cells. J. Immunol. 157, 1117-l 126.
42, Kagi, D., Lederman, B., Bilrki, K., Zinkernagel, R., and Hengartner, H. (1996)
Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in unmunological protection and pathogenesis m vivo. Ann. Rev. Immunol. 14,207-232
43. Cook, J. L., Potter, T. A., Bellgrau, D., and Routes, B. A. (1996) El A oncogene
expression m target cells induces cytolytic susceptibility at a post-recogmtion
stage in the interaction with ktller lymphocytes. Oncogene 12, 833-842.
44 Ljunggren, H -G. and Karre, K. (1986) Variations in MHC antigen expression on
tumours and its significance. Experimental strategtes and interpretations m the analysis of changes in MHC gene expression during tumour progression. Opposing influences of T cell and natural killer mediated resistance. J Zmmunogenet 13, 141-15 1.
45. Hoglund, P., Glas, R., Ohlen, C., Ljunggren, H.-G., and Karre, K. (1991) Alteration of the natural killer repertoire m H-2 transgenic mice: specificity of rapid
lymphoma cell clearance determined by the H-2 phenotype of the target. J Exp
Med. 174,327-334.
46. Hoglund, P., Ohlen, C., Carbone, E., Franksson, L., LJunggren, H.-G , Latour, A.,
Koller, B., and Karre, K. (1991) Recognition of b2-microglobulin-negative
(b2m-)
T-cell blasts by natural killer cells from normal but not from b2m- mace*
nonresponsiveness controlled by b2m- bone marrow in chimeric mice. Proc. Nat1
Acad SCL USA 88,10332-10336.
47. Liao, N.-S., Bix, M., Zijlstra, M., Jaenisch,R, and Raulet, D. (1991) MIX classI deficiency
susceptibility to natural killer (AK) cells and impaned NK activity. Scrence 253,199.
48. Daniels, B. F., Karlhofer, W. E., Seamen, W. E., and Yokoyama, W. M. (1994) A
natural killer cell receptor specific for a major histocompatibtlity complex class I
molecule. J. Exp. Med. 180,687-692.
141
49. Schrier, P. I., Bernards, R., Vaessen, R. T. M. J., Houweling, A., and van der Eb,
A. J. (1983) Expression of class I major histocompatibility
antigens switched off
by highly oncogenic adenovirus 12 in transformed cells. Nature 305, 77 l-775.
50. Mellow, G. H., Fiihring, B., Dougherty, J., Gallimore, P. H., and Raska, K., Jr.
(1984) Tumorigenicity of adenovirus-transformed rat cells and expression of class
I major histocompatibihty antigen. Virology 134,46OA65.
51. Haddada, H., Sogn, J. A., Coligan, J. E., Carbone, M., Dixon, K., Levine, A. S.,
and Lewis, A. M., Jr. (1988) Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicrty
of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian
hamster cells. J. Virol. 62,2755-2761.
52. Haddada, H., Lewis, A. M., Jr., Sogn, J. A., Coligan, J. E., Cook, J. L., Walker, T. A.,
and Levine, A. S. ( 1986) Tumorigenicity of hamster and mouse cells transformed by
adenovirus types 2 and 5 is not influenced by the level of class I major histocompatibility antigens expressed on the cells. Proc. Natl. Acad. Sci. USA 83,9684-9688.
53. Chadwick, B S. and Miller, R. G. (1992) Hybrid resistance in vitro. Possible role
of both class I MHC and self peptides in determining the level of target cell sensitivity. J Immunol 1458,2307-23 13.
54. Brutkiewicz, R. R. and Welsh, R. M. (1995) Major h&compatibility
complex class I
antigens and the control of viral mfections by natural killer cells. J. Viol. 69,3967-397 1.
55. Kranz, C. K., Routes, B. A,, Quinlan, M. A., and Cook, J. L. (1996) El A second
exon requirements for induction of target cell susceptibility to lysis by natural
killer cells. Virology 217,23-32.
56. Bukowski, J. F. and Welsh, R. M. (1985) Inability of interferon to protect virusinfected cells against lysis by natural killer (NK) cells correlates with NK cellmediated antiviral effects in vivo. J. Immunol. 135,3537-3541.
57. Trinchieri, G., Granato, D., and Perussia, B. (1981) Interferon-induced resistance
of tibroblasts to cytolysis mediated by natural killer cells: specificity and mechanism. J. Immunol 126,335-340.
58. Piontek, G. E., Taniguchi, K., Ljunggren, H.-G., Gronberg, A., Kiessling, R.,
Klem, G., and Karre, K. (1985) YAC-1 MHC class I variants reveal an association between decreased NK sensitivity and increased H-2 expression after interferon treatment or in vivo passage. J. Immunol. 135,4281-4288.
59 Routes, J. M. (1992) Interferon increases class I MHC Ag expression on adenovirus infected human cells without inducing resistance to NK cell killing, J,
Immunol. 149,2372-2377.
60. Routes, J. M. (1993) Adenovnus ElA inhibits IFN-induced resistance to cytolysis by natural killer cells. J. Zmmunol. 150,43 15-4322.
61. Routes, J. M., Li, H., Bayley, S. T., Ryan, S., and Klemm, D. J. (1996) Inhibition
of IFN-stimulated gene expression and IFN induction of cytolytic rerstance correlate with ElA-~300 binding. J. Immunol. 156, 1055-1061.
62. Pross, H F., Baines, H. T., Rubin, P., Shragge, P., and Patterson, M. S. (1981)
Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells.
IX. The quantition of natural killer activity. J Clin. Immunol. 1, 5 l-63.
12
Determination
of Adenovirus
of the Transforming
Oncogenes
Activities
Introduction
143
144
2. Materials
1. Baby rats: A litter of (approx S-12 pups) 2-d-old neonatal Fisher rats can be
purchased from Harlan-Sprague Dawley (Indlanapohs, IN).
2. Nude mice (Harlan-Sprague Dawley).
3. Plasmids: ElA 12s and T24 ras plasmlds can be obtained from the authors
as gift.
4. Carrier salmon sperm DNA (Sigma, St. Louis, MO). Purify by phenol extraction.
5. Phosphate-buffered saline (PBS) without Ca2+ and Mg2+: 8.0 g NaCl, 0.20 g KCl,
1.15 g Na2HP04, 0.2 g KH2P04. Dissolve in 900 mL triple-distilled water and
adjust the pH to 7.2 with HCl. Make up the volume to 1.OL, sterilize by autoclaving and store at 4C.
6. Dispase-collagenase: Dissolve 250 mg of dispase II and 25 mg of collagenase H
(Boehringer Mannheim, Indianapolis, IN) in 100 mL of PBS by stirring with a
magnetic bar for 30 min at room temperature. Filter sterilize and store at -20C.
7 2X HeBS: 8.182 g NaCl, 5,958 g HEPES, 107 mg Na,HP04 Dissolve the above
chemicals in 400 mL of water and adjust the pH to 7.10 + 0.05. Make up the
volume to 500 rnL, filter sterilize, and store at -20C.
8. 2 MCaC12: Dissolve 22.2 g of calcium chloride (CaC12*7H20, Sigma) in 100 mL
of water, filter sterilize, and store at -20%
9. G418 stock: Prepare 10 mg/mL active ingredient of G418 (Gibco-BRL,
Gaithersburg, MD; concentration of the active ingredient varies with the lot number) in 100 miVHEPES, pH 7.3, filter sterilize, and store at -20C m the dark.
10. 10X Giemsa: Dissolve 3 g of Giemsa (Sigma) in 250 mL of glycerol by stirring at
37C overnight. Next day, add 250 mL of methanol and mix well. Store the solution in dark for 2 wk. Filter through one layer of paper towel and store at room
temperature in the dark.
3. Methods
3.1. Preparation
of BRK Cells
145
Fig. 1. Illustration of the rat pup with the skin removed on the dorsal side (A) and
the site of incision of the muscle in between ribs and lumbo-sacral region exposing the
kidney (B).
2.
3.
4.
5.
6.
7.
8.
9.
the ribs and lumbo-sacral region and apply pressure with a forceps. The kidney
pops out as shown in Fig. 1B. Remove kidneys to a sterile bacterial Petri dish
containmg 20 mL PBS without Ca*+ and Mg*+ (see Note 2).
Wash the kidneys three times with PBS and transfer to a fresh bacterial Petri dish
containing PBS.
Remove capsules and the attached blood vessels with pointed forceps.
Transfer kidneys to a new bacterial dish and mince into small pieces (approx 0. I0.2 mm in diameter) with scissors.
Add dispase-collagenase (50 mL/20 kidneys) and transfer to a 125-mL Erlenmeyer flask (see Note 3).
Add a sterile magnetic stirring bar and stir vigorously for 10 min at 37C.
Wait for 2 min to allow the clumps to settle, and transfer the supernatant to a
50 mL sterile centrifuge tube kept in an ice/water bucket.
Add 50 mL of dispase-collagenase to the clumps and stir again until the clumps
are dispersed (approx 10 min).
Pool the supernatant and the second digest and centrifuge for 5 mm at 200g
and 4C.
146
10. Remove the supernatant and suspend the cells in a small volume (50 mL) of
prewarmed (37C) DMEM containing 10% fetal bovine serum. Dilute to 1 L
(20 kidneys).
11. Disperse the cells with the help of lo-mL plpet attached to a mechanical pipettmg
devise and dispense 5 mL to each 25-cm2 tissue-culture flask (or 60-mm tissueculture dish) and incubate at 37C in a CO, incubator overnight. Cells can be
transfected with DNA, 16-24 h after plating.
147
The immortalized and transformed colonies of p12S (243R) and two mutants
of 12s with and without T24 ras are shown in Fig. 2.
3.4. Characterization
of Transformed Cells
To determine the extent of transformation, the transformed cells are generally assayed for their ability to form anchorage-independent colonies on semisolid (soft-agar) media in vitro or examined for their ability to form tumors m
athymic mice or syngeneic hosts.
3.4.1. Soft-Agar-Colony
Assay
3.4.2. Tumorigenesis
in A thymic Mice
of trans-
148
149
such as lungs. If the burden of primary tumors is heavy for the animals, the
primary tumors have to be surgically removed and animals are maintained for
additional 2-4 wk and autopsied.
4. Notes
1. Neonatal Fisher rat pups should be used between 2 and 3 d after birth. If older
pups are used, the efficiency of transformation may be lower.
2. During preparation of BRK cells, the capsule present in the kidney should be
removed as much as possible with a tine-pointed forceps. If it is not fully
removed, the preparation will contain flbroblasts. The fibroblasts will grow faster
than epithelial cells and will not die with 50 pg/mL of G4 18 (normal concentration for BRK cells); this will result in background colomes.
3. The minced kidneys are digested with dispase-collagenase for a maximum period
of 15 min each time. Digestion for longer period of time will reduce the viability
of epithelial cells.
References
1. Moran, E. (1993) Interactton of adenovirus proteins with pRb and ~53. FASEB J.
7,880-885.
2. Nevins, J. R. (1995) Adenovuus ElA: transcription regulation and alteration of
cell growth control. Curr. Topics Microbial. Immunol. 199,25-32.
3. Harlow, E. (1996) A research shortcut from a common cold virus to human cancer. Cancer 78,558-565.
4. Ruley, H. E. (1983) Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature 304,602-606.
5. Zerler, B., Roberts, R. J., Mathews, M. B., and Moran, E. (1987) Different functional domains of the adenovirus ElA gene are involved in regulation of host cell
cycle products. Mol. Cell. Blol. 7, 821-829.
6. Chinnadurai, G. (1992) Adenovirus Ela as a tumor-suppressor gene. Oncogene 7,
1255-1258.
7. Mymryk, J. S. (1996) Tumor suppressive properties of the adenovirus 5 Ela
oncogene. Oncogene 13,1581-1589.
8. Sussenbach, J. S. (1984) The structure of the genome, in The adenoviruses
(Ginsberg, H. S., ed.), pp. 35-124. Plenum, New York.
9. Kuppuswamy, M. and Chinnadurai, G. (1988) Cell type dependent transformation by adenovirus 5 Ela proteins. Oncogene 2,567-572.
10. Graham, F. L. and Van der Eb, A. J. (1973) A new technique for the assay of
infectivity of human adenovirus 5 DNA. Virology 52,456-467.
11. Wigler, M., Pellicer A., Silverstem S., Axe1 R., Urlaub G., and Chasm L. (1979)
DNA-ediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc. Natl. Acad. Sci. USA 76, 1373-1376.
12. Kuppuswamy, M., Subramanian, T., and Chinnadurai, G. (1988) Separation of
immortalization and T24-ras oncogene cooperative functions of adenovirus El a.
Oncogene 2,613-615.
13
Cell Survival and Apoptosis Assays
for ElB 19K and B&2 Family Members
T. Subramanian
and G. Chinnadurai
1. Introduction
Programmed cell death, or apoptosts, is an important event in the normal
development and homeostasis of multicellular organisms (2). Apoptosts is
regulated by a number of genes that promote or suppress cell death. The bcl-2
family of genes constitute important regulators of apoptosrs. Several members
of the bcl-2 family such as bcl-2 (2) and bcl-xL (3) suppress apoptosts, whereas
certain other members such as bax (4), bak (5), and bzk (6) promote apoptosts
Certain animal viruses such as adenovirus, Epstein-Barr vuus, herpesvuus
stmarri, and African swine fever virus also encode structural and functional
homologs of Bcl-2. The methods currently available to determine the anti- or
pro-apoptotic activrttes of various cellular and viral Bcl-2 homologs are cumbersome. Here, we describe highly reproducible, simple methods that we have
developed to assaythe activities of adenovirus E I B 19 kDa, Bcl-2, and various
pro-apoptotic interacting partners.
2. Materials
1. Primary baby-rat kidney cells. prepare thesecells from 2-d-old Fisher baby-rat
kidneys (BRK) according to the procedure described by Subramanian and
Chinnadurai (Chapter 12).
2. BRK ElA/p53V135 cell lme purchase from Scintech, St LOUIS, MO, or prepare
from BRK cells.
3. Plasmrds: obtain p12S 177-9+ras from the authors as gift.
757
752
3. Methods
3.1. Antiapoptosis
Assay
This assay is based on results obtained by Oren and colleagues (7), who
showed that certain hematopoietic cells expressing a temperature-sensitive
mutant of the p53 tumor suppressor protein (p53tsVall35) undergo rapid and
complete apoptosis at the permissrve temperature (32.5C) at which the p53
protein assumes wild-type conformation. These results were further extended
by Debbas and White (81 who showed that BRK cells transformed with adenovirus Ela and p53tsVall35 grow normally at 38.5C and undergo rapid
apoptosis at 32.5OC. BRK cells expressing Ela and p53tsVa1135 offer two
advantages: They can be readily transfected with various test genes by the conventional calcmm-phosphate method, and the antrapoptotrc activrty of various
test genes can then be assayed by simple temperature
assay to determme the antiapoptotic activity of adenovirus ElB 19-kDa protein (9) and a number of Bcl-2 family protems (10).
In order to determine the apoptotic activity of various pro-apoptotic genes, a
transient transfection procedure is commonly used. In this assay, the proapoptotic genes are cotransfected with the Escherzchia coli 1acZ reporter gene.
Among the transfected cells (identified by X-gal staining), the cells undergoing apoptosis are identified by microscoptc exammation. The apoptotic and
nonapoptotic cells are discriminated by virtue of a rounded (dead) or flat (live)
morphology (II). However, we and others have found that this method is laborious and can sometimes yield unreliable results. We have developed a sample
colony assay that we have used to determine the anttapoptotic activity of vartous Bcl-2 protein family members such as Bax, Bak, and Bik. In thts chapter,
the assays for evaluating
anti or pro-apoptotic
activity
are descrtbed.
153
We have observed under these assay conditions, cells expressmg antiapoptosis proteins such as E 1B 19 kDa or Bcl-2 retain 60-l 00% vlabllity over
a period of 96 h. In contrast, cells transfected with the vector lose near total
vlabllity within 72 h (Fig. 1).
3.2. Apoptosis Assay for Bcl-2 Family Members
This method can be used to assay the activity of pro-apoptotlc members of
the Bcl-2 family or their mutants. The assaymethod is based on the cotransformation of primary BRK cells with a Ela super-transforming mutant (containmg a deletion mutation m the second exon) and T24 ras oncogene. We have
observed that cotransfectlon of the various death-inducing members of the
Bcl-2 family consistently mhlbit transformed-colony formation and specific mutations that abolish the death-promoting activity relieve this inhibltton. In this assay, BRK cells are transfected with a plasmid (p12S177-9+ras)
that expresses both the E 1 a mutant (12s 177-9; ref. 12) and the T24 ras
oncogene along with the plasmid (pcDNA3 or pRcCMV-based) that expresses
the pro-apoptotlc gene and the dominant selection marker neo. Plasmids
expressing Bax, Bak, or Blk are used as the posltlve
154
Subramanian
and Chinnadurai
20
18
16
14
12
IO
08
06
04
02
00
0
24
48
72
hours
n , bcl-2,
a, @l-l, V, BHRFI
ing 10% fetal calf serum (use four flasks for each experiment) 1 d prior to
transfection
2 Transfect 1 pg of p 12s 177-9+ras and equimolar amounts of plasmtds expressmg the death-mducmg genes or the vector by the standard calcium-phosphate method.
3. Twenty-four hours after transfectton, add G418 (50 I.lg/mL) and select the cells
for 10 d with three changes of medmm
4 Stain the transformed colontes with Giemsa, count for quantitation,
and
take photographs
A cell death assaywas performed for bc12 family genes m BRK cells and the
results are presented in Fig. 2.
4. Notes
1 It is preferable to use the BRK-ElA/p53V135
cell hne m early passages Cells at
more than passage SIX ~111 produce spontaneous resistance at 32 5C. In most
experiments we use cells m the fourth passage. The early-passage cells will die at
a much faster rate at 32 5C compared to cells from a higher passage.
2 The optimum concentrations of G418 are 50 and 300 ,ug/mL for prtmary and
established BRK cells, respectively.
155
pcDNA3
Bik
Bax
Bak
Bcl-xS
Bcl-2
Bcl-xL
BikAGD
80 -I
I
E
p 60
s5
2E 402
20 -
pcDNA3
Bik
BikAGD
Bax
Bak
Bcl-xS
Bcl-2
Bcl-XL
156
Subramanian
and Chinnadurar
References
1. Elhs, R E., Yuan, J J , and Horvttz, H. R. (1991) Mechanisms and functions of
cell death Annu Rev. Cell Brol 7,663-698.
2 Korsmeyer, S J. (1992) B&2 mitrates a new category of oncogenes: regulators of
cell death. Blood 80, 879-886.
3 Boise, L. H., Gonzalez-Garcia, M., Postema, C E., Dmg, L , Lindsten, T , Turka
L. A., Mao, X., Nunez G , and Thompson, C. B. (1993) B&x, a bcl-2- related
gene that functrons as a dominant regulator of apoptotrc cell death. Cell 74,
597-608.
4. Oltvar, Z. N., Mtlhman, C. L , and Korsmeyer, S J (1993) Bcl-2 heterodlmertzes
in VIVO with a conserved homolog, Bax, that accelerates programmed cell death
Cell 74,609-619.
5 Chrttenden, T., Flemmgton, C., Houghton, A B , Ebb, R. G., Gallo, G J.,
Elangovan, B., Chmnadural G., and Lutz R. J (1995) A conserved domain m Bak,
distinct from BHl and BH2, mediates cell death and protem bmdmg functions
EMBO J 14,5589-5596
6 Boyd, J M., Gallo, G. J , Elangovan, B , Houghton, A B., Malstrom, S , Avery,
B J., Ebb, R. G , Subramaman T., Chtttenden, T., Lutz, R J., and Chmnadurat
G. (1995) Brk, a novel death-inducing protein with a distinct sequence motif,
interacts with viral and cellular survrval promotmg protems. Oncogene II,
1921-1928.
7 Mlchalovttz, D., Halevy, O., and Oren, M. (1990) Condrtional mhrbmon of transformatron and of cell prohferation by a temperature-sensitrve mutant of p53. Cell
62,67 l-680.
8 Debbas, M. and White, E (1993) Wild-type ~53 mediates apoptosls by El A,
which is inhibited by ElB. Genes Dev 7,546554
9 Subramanian, T., Boyd, J M , and Chmnadurai, G (1995) Functronal substrtutton
Identifies a cell survival promoting domain common to adenovnus E 1B 19 kDa
and Bcl-2 proteins. Oncogene 11,2403-2409
10. DSa-Eipper, C., Subramanian, T., and Chinnadurat, G (1996) Bfl-I, a bcl-2
homologue, suppresses p53-induced apoptosrs and exhibrts potent cooperatrve
transforming activity. Cancer Res 56,3879-3882.
11. Miura, M , Zhu, H., Rotello, R., Hartwerg, E. A., and Yuan, J Y. (1993) Inductton
of apoptosis m tibroblasts by IL-1 beta-converting enzyme, a mammalian homologue of the C. elegans cell death gene ted-3 Cell 75,653-660
12. Subramanian, T., La Regina M., and Chinnadural, G (1989) Enhanced ras
oncogene mediated cell transformatton and tumortgenesrs by adenovrrus 2
mutants lacking the C-termmal region of E la protein. Oncogene 4,4 15-420
14
In Vitro Transcription
Probing the Molecular Functions
of Adenovirus Regulatory Proteins
Paul M. Loewenstein,
Chao-Zhong
1. Introduction
Adenoviruses (Ads), like other DNA tumor viruses, have evolved specific
regulatory
virus replication
by controlling
the transcrip-
tion of other viral genes as well as that of key cellular genes that regulate cellcycle progression and cellular DNA synthesis. Of parttcular interest is the Ad
ElA transcription unit that encodes two major proteins of 243 and 289 amino
acids. The 28913 protein is identical to the 243R protein except that tt also
contains conserved region 3 (CR3, restdues 140-188), a powerful transcriptional activator of Ad early genes (see ref. I for review). The 243R protem
encodes diverse biological functions, including the ability to induce cell-cycle
progression, immortalize cells, transform cells in cooperation wtth other
oncogenes, and paradoxically to inhibit tumorigenicity and cell differentiation
(I). These El A 243R functions are encoded within multiple protein domains
that can transcriptionally acttvate or repress cellular genes involved in the regulation of cell proliferation and cell differentiation.
Recently, in vitro proteipprotein binding studies have revealed that viral
regulatory proteins such as ElA can interact with sequence-specific transcriptional activators as well as with several general transcription factors (GTFs) of
the cellular transcription machinery. The significance of these protein-protein
interactions in transcriptional regulation can be addressed mechanistically by
the applrcatton of the vitro transcription methodology using purified recombinant viral proteins.
From
757
and Protocols
NJ
158
Transcription m mammalian cells by RNA polymerase II IS a complex process that mvolves the formation of a huge premitlation complex composed of
at least 50 different polypeptides that can be classified into several groups A
first group of polypeptides constitutes the general transcription machinery and
includes RNA polymerase II and the general transcrrptron factors TFIIA,
TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (see ref. 2 for review). A second group
consists of nucleic acid sequence-specific transcriptional activators that stimulate transcrtption, at least in part, by mcreasing the number of functional transcription complexes (see ref. 3 and references therein). A third group conststs
of a growing number of proteins classified by function as coactivators, positive
cofactors, negative cofactors, and general repressors of transcription (2). This
third group activates or represses transcription through protein-protein interactrons wrth transcription factors, often components of the general transcrlptton machmery. The E 1A protems do not bind to specific DNA sequences and
then transcriptional activities best fit mto the third group of transcriptton factors that function through protein-protein interaction.
The molecular mechanism of transcriptional regulation by viral protein
domains can be effectively explored using in vitro transcription systems Of
particular importance has been the development of a procedure for preparing
extracts from nuclei of HeLa cells that directs accurate transcription mitiation
m vitro from RNA polymerase II promoters, as described by Dignam et al. (4).
Transcriptionally active nuclear extracts can be prepared from different cell
types and from as few as 3 x lo7 cells (5). The preparation of transcription
extracts and the assay conditions are often modified to optimize transcription
of specific genes (see Note 1). Of additional value has been the use of proteinaffimty and antibody-depletion experiments to define components within
nuclear extracts that interact with specific viral or cellular proteins and are
involved in transcriptional regulation. Fmally, given the clonmg and expression of most of the transcription factors that comprise the general transcription
machinery, it is possible to analyze the functions of viral proteins in a defined
reconstituted transcription system. Such studies hold promise of a detailed
molecular understanding of the interaction of viral regulatory protems with the
transcriptional machinery of the cells. Examples of the use of in vitro transcription to analyze Ad regulatory functrons are described below.
7.7. Using in Vitro Transcription
to Demonstrate the Autonomous
Transactivation
Activity of Ad ElA Conserved Domain 3 (CR3)
Analysis of E 1A mutants by transient expression has demonstrated that CR3
IS essential for transactivation of early viral genes by ElA 289R (for review,
see ref. I). To determine whether CR3 is sufficient for transactivatton, a 49ammo acid peptide encodmg CR3 plus 3 ammo acids m exon 2 (termed PD3)
PD3
159
-
E3-
1 2
Fig. 1. In vitro transcription of the Ad2 E3 promoter and the Ad2 MLP in the presence (+) and absence (-) of PD3 peptide. In vitro transcription was performed with
reaction mixtures containing E3 or MLP plasmids as templates and subjected to primer
extension analysis. Primers consisted of 5 end-labeled synthetic oligonucleotides
complementary to the E3 mRNA (positions + 108 to + 137) or MLP mRNA (positions
+67 to +86) from the start site of transcription. Labeled cDNA products were analyzed
by electrophoresis on a 6% polyacrylamide-7 M urea gel.
was chemically synthesized and tested for its ability to transactivate Ad promoters in vitro. In vitro transcription products were analyzed by primer-extension analysis to ensure that transcription initiated at the correct start site. PD3
(100-500 ng) added to a reaction mixture containing 500 ng of DNA template,
either the Ad early-region
3 (E3) promoter or the Ad major-late promoter
(MLP), stimulated transcription 5- to 20-fold (see Fig. 1 for a typical analysis).
These results directly demonstrate that the sequences within CR3 are responsible for and sufficient for ElA transactivation (6). PD3 is the smallest known
autonomous transactivator.
(Ad l-80)
Analysis of E 1A mutants by transient expression indicated that the transcriptional repression function of ElA 243R requires sequences within the
nonconserved N-terminal 40 amino acids, within CR1 , and also within CR2 in
some studies (for review, see ref. 1). To study the mechanism of the poorly
understood ElA repression function, it was important to establish a system
that faithfully recapitulates ElA repression in vitro. This would permit a biochemical definition of the viral and cellular components involved in the ElA
repression reaction. A recombinant protein containing only the ElA 80 N-terminal amino acids (El A l-SO), which includes the N-terminus plus all of CR1 ,
and a series of ElA l-80 deletion-mutant
proteins were prepared. These pro-
160
ICE-CAT
Fig. 2. Transcription repression m vitro of the insulm II promoter by purtfied
recombmant E 1A l-80 protem pICE-CAT was used as template for the m vitro transcrtption reaction Transcrrpts were analyzed by prtmer extension using a CAT primer
Reaction mrxtures contained from 0 to 1000 ng of ElA l-80, as indicated
repression of promoters
prevt-
Adenovirus
161
Regulatory Proteins
I;
tii+
2
+
!i!i
2
-8
$h
H
53 -0
2
-cl
? % @
h h z;
zi iii c
HIV LTR -
123456
Fig. 3. TBP can restore transcriptional activity to an ElA l-SO-depleted nuclear
extract. The transcriptional activity of the original nuclear extract (NE) (lane 1) is
repressed by added ElA l-80 protein (400 ng, lane 2). Transcriptional
activity
is lost by passage through an ElA l-80 affinity column (lane 4), but not an ElA
l-8084-25 column (lane 3). The addition of TBP (5 ng, lane 5) restores transcriptional activity to the ElA I-80 affinity depleted nuclear extract. The activity restored
by TBP can be repressed by addition of E 1A l-80 (400 ng, lane 6). In vitro transcription and primer extension analysis was performed with pBennCAT (HIV LTR) as
template and a CAT primer.
from the nuclear extract. Western analysis demonstrated that the general transcription factor, TBP (TFIID), was depleted from the extract and was bound to
the E 1A l-80 affinity column (8). Significantly, activity of the depleted extract
was completely restored by addition of TBP, thus providing strong evidence
that TBP (TFIID) is a target of ElA repression (see Fig. 3).
2. Materials
To minimize RNase contamination, all reagents should be made with DEPCtreated water: add 1 mL of DEPC per liter of water, shake well, and autoclave
after 1 h. Wear gloves during all operations to avoid contamination with finger
RNases. Pipetting devices should be wiped down with ethanol and never used
with solutions containing RNase. All reagents can be stored at -20C.
162
loewenstein,
3. Methods
3.7. Preparation of the Nuclear Transcription
Extract
Nuclear extracts can be made from vutually any volume of cells grown m
monolayer or in suspension culture (5). For reproducrbihty and convenience,
the majority of nuclear extracts are from liter quantities of HeLa cell-suspension culture. The protocol used in our laboratory IS a modtficatron of that of
Dtgnam et al. (4). We use monolayer cultures of HeLa cells obtained from
Adenovirus
Regulatory Protems
163
of In Vitro Transcription
by Primer Extension and by Run-Off Assay
3.2. Analysis
164
assay is the run-off assay that uses a DNA template cut with a restrlctlon
enzyme downstream of the transcriptlon start site. This simple solution creates
a site where the polymerase will fall off the template, thus terminating transcnptlon. By use of a 32P-labeled rNTP precursor, an RNA transcription product of specific length 1s synthesized and 1s resolved as a discrete band by
denaturing polyacrylamide gel electrophoresls and autoradiography. The second assay 1sprimer extension m which the RNA product of the in vitro transcription reaction 1s annealed with a 32P end-labeled deoxyohgonucleotlde
complementary to a discrete sequence downstream of the transcrlptlon start
site. Reverse transcription of the hybrid RNA product/DNA primer yields a
labeled DNA fragment of discrete length that spans the sequence between the 5
end of the primer and the transcription start site. This labeled cDNA fragment 1s
resolved by denaturing polyacrylamlde gel electrophoresls and autoradtography
The product of both assays can be quantitated by scanning densitometry or
PhosphorImage analysis (Molecular Dynamics, Sunnyvale, CA).
In our laboratory, we use primer extension almost exclusively. In general,
primer extension is more sensitive than run-off assay and permits a more accurate measurement of the transcription product from the authentic transcrlptlon
start site. For many of our studies, we use the CAT gene as a reporter for dlfferent promoters, thus allowing a single CAT primer to be used for a variety of
promoters. This also permits the use of multiple DNA-template promoters m a
single transcrtptlon reaction because the S-transcribed sequence from each
promoter 1susually sufficiently different m size and thus gives rise to primer
extension products of different lengths.
3.2.1. Preparatron of DNA Templates for In Vitro Transcription
Plasmld templates containing the promoter of interest are prepared by standard alkaline-lysis procedure or by use of commercial plasmid-preparation kits.
It is important, however, to further purify templates for in vitro transcrlptlon
analysis using phenol-chloroform extractlon followed by ethanol precipitation.
For primer extension, superhelical plasmid templates are used. For run-off
analysis, templates are linearized with a restriction enzyme m order to termlnate transcription at a known site.
3.2.2. Preparation of Radiolabeled
for Primer Extensron
Deoxyoligonucleotides
Adenovirus
Regulatory Proteins
165
When the transcrrption reaction IS complete, terminate the reaction by addition of 100 $ of stop mix. Then add 300 mL of 0.3 Msodium acetateand 300 &
of phenol/chloroform/isoamyl alcohol (50:50:2). Vortex the sample for 30 s
and separate the phases by centrrfugation at 10,OOOgfor 2 min. Transfer the
upper aqueous phase to a fresh tube containing 1 mL of ethanol, mtx, and place
on dry ice for 15 min. Centrifuge the sample at 10,OOOgfor 10 min at 4C, rinse
the pellet carefully with 500 + of 80% ethanol (-2OC), and briefly dry the
pellet containing the RNA product in a vacuum desiccator.
3.2.3.3.
PRIMER-EXTENSION ANALYSIS
166
Model V16). The smaller plate (facing the apparatus) is silicomzed with RamX to facilitate separation of the plates after electrophoresis. To prepare the gel,
stir with a magnetic bar 12 g of urea (Gibco-BRL ultrapure, RNase free) with
6.0 mL of 5X TBE, 4.5 mL of 40% acrylamidelbzs-acrylamrde (29: l), 300 pL
of 10% ammonium persulfate, and water to a final volume of 30 mL. When the
urea 1scompletely dissolved, add 20 l.iL of TEMED and ptpet the mixture into
an assembled gel sandwich held at about a 40 angle. Insert a 15well comb
and the allow the gel to polymerize in a horizontal position for 3 h or overnight. Prior to loading the samples, mount the gel on the electrophoresis chamber, add TBE to the upper and lower chambers, and clean the gel teeth by
pipetting TBE up and down. Pre-electrophorese the gel for approx 15 min at
400 V constant. Load the samples (10-20 &) and a labeled size marker (e.g.,
a 50-bp ladder. see Note 2) mto mdividual lanes on the gel and electrophorese
at 400 V constant until the bromophenol blue marker migrates to the bottom
of the gel.
In our laboratory, the gel is pulled onto 3MM paper. Briefly separate the gel
sandwich by gentle prying with a fine-bladed spatula. The gel will adhere to
the larger, unsiliconized plate, Put the gel-containing plate flat on a bench top
and carefully position a sheet of 3MM paper over the gel. Rub the paper gently
but firmly and carefully pull off the paper with the adherent gel. The gel can
then be drred on a gel dryer prior to autoradiography or simply covered with
plastrc wrap and autoradiographed with an mtensifymg screen at -70C overmght. For quantitation of signals by PhosphorImage analysis, gels are dried
and exposed to a phosphor storage screen.
3.2.4. In Vitro Transcription Using the Run-Off Assay
The transcription reaction for the run-off assay is virtually the same as that
described m Subheading 3.2.3.1. with the following modifications. First, the
DNA template is cut at a convenient sue with a restriction enzyme. Second, the
10X rNTP mixture consists of 5 mM ATP, GTP, and CTP, but only 0.25 rm?4
UTP. Third, 32P-UTP (0.5 & of 10 mCi/mL, 800-1000 Ci/mmol) is added to
the reaction mixture. Finally, RNA is isolated as described m Subheading
3.2.3.2. and is directly analyzed by gel electrophoresis as described in Subheading 3.2.3.4.
4. Notes
1, When one ISstudying transcriptional regulation in vitro by the addition of a candidate regulatory protein to a transcnptlon-reaction mixture, it 1s important that
the transcription signal in the absence of added regulatory protein be optimized
In the case of transcripttonal activation, it 1s desirable to use conditions that are
suboptimal in the absence of added activator so that transactwatlon can be easdy
Adenovirus
Regulatory Protems
167
measured over a wide dynamic range. By contrast, when one IS studymg transcription repression, it IS desirable to use condmons that provide a strong transcription signal, thus providing a sensitive assay for measuring repression by
added protein These goals can often be achieved empirically by one of two
means. First, the amount of nuclear extract in the reaction mixture can often be
titrated to provide the desired level of transcription m the absence of added protem factor. Second, it is possible to prepare extracts that possess innately high or
low activity by altering the ionic strength of the salt used to prepare the nuclear
extract. These extracts may contain greater or lesser amounts of transcription
factors or cellular inhibitors of transcription, some of which may represent a cellular target of the added regulator protein The putative cellular target may not be
rate-limiting m the nuclear-transcription extract prepared under standard conditions. Specific modification of the reaction mixture can also achieve the desired
results For example, to demonstrate in vitro transactivation of the HIV LTR
promoter by the HIV- 1 Tat transactivator protein in run-off assays, it was necessary to reduce the basal transcription level of extracts by the addition of 6 mM
sodmm citrate (9).
2. As a size marker for analysis of transcription products, it is convenient to label
50 or 100 bp DNA ladder as described by the manufacturer (Gibco-BRL)
Acknowledgments
We thank Carolyn E. Mulhall for editorial assistance. The work from the
authors laboratories was supported by Research Career Award AI-04739 and
Public Health Service grants CA-29561, CA-54703, and AI-28201 to M. G
from the National Institutes of Health.
References
1 Shenk, T (1996) Adenoviridae. the viruses and then replication, m Virology
(Fields, B. et al , eds ), Lippincott-Raven, New York, pp 2111-2148
2. Zawel, L and Reinberg, D. (1995) Common themesm assemblyand function of
eukaryotic transcription complexes. Ann Rev. Bzochem 64,533-561
3 Choy, B. and Green, M. R. (1993) Eukaryotic activators function during multiple
steps of premitation complex assembly. Nature 366, 53 l-536.
4. Dignam, J. D , Lebovitz, R M., and Roeder, R. G. (1983) Accurate transcription
initiation by RNA polymerase II m a soluble extract from isolated mammalian
nuclei. NucIelc Aczds Res 11, 1475-1489.
5. Lee, K A W and Green, M R. (1990) Small-scale preparation of extracts from
radtolabeled cells efficient in pre-mRNA splicing. Methods Enzymol 181,20-30.
6. Green, M , Loenstein, P M , Pustazi, R., and Symington, J. S (1988) An adenovirus EIA protein domain activates transcription in vzvo and In vztro m the absense
of protein synthesis, Cell 53, 92 l-926.
7. Song, C-Z , Tierney, C. J , Loewenstem, P. M., Pusztai, R., Symmgton, J S.,
Tang, Q , Toth, K., Nishikawa, A , Bayley, S. T., and Green, M. (1995) Transcnp-
168
Loewenstein,
15
Cell Microinjection
in Vivo Analysis of the Functional Domains
of Viral Regulatory Proteins
Maurice Green, Andrew Thorburn,
1. Introduction
Mammalian-cell microinjection is a powerful method for analyzing the in
vivo functions of viral genes and viral gene products. By microinjection, a
controlled amount (ranging from 1 to many thousands of copies) of a viral or
cellular gene, a protein product of a gene, a polypeptide fragment encoding a
specific protein domain, or an RNA molecule can be delivered into a target cell
and the functional consequences analyzed. Injection of DNA into the nuclei of
cultured mammalian cells provides a sensitive bioassay for gene expression.
The product of a single injected gene copy can be detected using standard
immunofluorescent, immunochemical, and autoradiographic techniques (I).
The direct analysis of protein function by microinjection has been facilitated
by the ability to produce biologically active recombinant proteins and protein
fragments encoded by many interesting genes.
A valuable use of microinjection is to deliver antibody targeted to a specific protein domain in order to analyze the requirement of the protein for specific cell functions, for example, cell-cycle progression, transcription of
specific genes, or intracellular transport. Antibodies introduced by microinjection into mammalian cells are highly specifk and nontoxic. Several additional
cell-microinjection strategies have been used successfully. These include, for
example, the delivery and functional analysis of antisense RNA and the determination of morphological alterations in response to an injected molecule,
including cell transformation. Cell microinjection has been uniquely used to
address mechanistic questions, such as whether the function of a microinjected
From
169
and Protocols
NJ
170
Cell Microinjection
Ad2
E2 Early
Gene
E2 Protein
Ad2
El A Gene
or
Protein/
Protein Domain
lmmunoRuorercence
tive models was designed. The reporter pE2-CAT (the CAT gene driven by the
promoter of Ad E2) was coinjected with the PD3 peptide in the presence or
absence of the protein synthesis inhibitor
(cycloheximide
or antsomycm),
and
the formation of the CAT RNA product measured by in situ hybridization with
an 35S-labeled CAT DNA probe (see Fig. 2 for schematic). Under conditions
in which protein synthesis was effectively blocked, PD3 efficiently activated
the E2 gene resulting in the accumulation of E2 RNA (see Fig. 3). Thus cell
microinjection uniquely demonstrated that the E 1A CR3 domain transactrvates
early viral genes by a direct mechanism involving interaction with a pre-existing cellular factor(s) (4).
1.1.3. Microinjection of an Ad EIA Protein-Fragment
to Analyze E 1A Transcriptional Repression-immune
fluorescence
Analysis of the Protein Product of a Repressed Gene
172
pE2CAT
ElA PD3
tmnsoctivator
6 hr
Cell
Cycloheximide
Or
Amsomycm
Fii
Cells
Hybridization
with Wobeled
CAT DNA
Automdiogrophy
Fig. 2. Cell microinjection in situ hybridization assay for gene activation in the
absence of protein synthesis. The schematic shows the assay for activation of the E2
gene fused to the CAT reporter (pE2CAT) by the ElA PD3 (protein domain 3) transcriptional activator in the presence of the protein-synthesis inhibitor cycloheximide
or anisomycin. Expression of the CAT gene is measured by in situ hybridization followed by autoradiography.
-CH
+CH
173
Cell Microinjection
SW0
Ear
Gene
Ad2 El A Gene
SV40
large
or
Protein/
PrWein Domain
1 Immunofluorexence
174
T Antigen
Marker
pl-11
Cell Microinjection
175
Protein/
Protein Domain
w6
or
Oncogene
J-
Microinjection
BudR
20-24
hr
BudR lmmunofluorescence
to overcome the blockage caused by Ras antibody, indicating that EIA can
substitute for Ras and may act downstream of Rus in the cell cycle (IO). The
temporal requirements for several cell proliferation-related genes in addition
to ras, including fos, $%I, and fru-2 (11,12), have been delineated by the
microinjection of specific antibodies at various times during the cell cycle.
Thus microinjection of specific antibodies facilitates analysis of complex cellular regulatory functions.
1.1.6. Microinjection of Specific Antibody to Analyze the Role
of a Putative Coactivator in the Transcription of Coinjected Promoter/
IacZ Repotter Constructs-Calorimetric
Assay for IacZ-Expressing Cells
The El A 243R protein encodes functions required for the induction of cellular division and the inhibition of cell differentiation. The N-terminal region
of E 1A can interact with the p300KBP (CREB-binding protein) family of proteins that are believed to function as coactivators for the transcription of genes
in pathways leading to the inhibition of cell division and the promotion of cell
differentiation. The transcription factor CREB, which is a component of the
176
2. Materials
2.7. Cell Culture for Microinjection
For microinjection experiments,standardcell-culture techniquesare usedto maintain stock cultures of cells, generally in Dulbeccos modified Eagles medium
(DMEM) supplementedwith the appropriate serum,either fetal bovine serum (FBS)
or calf serum (CS). Cells aretrypsinized and plated into 35-mm culture dishes containing cover slips marked to facilitate the location where cells are to be injected.
Imprinted cover slips are available commercially. For example, Eppendorf
(Westbury, NY) makes 12-mm circular cover slips (CELLocate 5245) that contain a
pattern of labeled 175- or 55-mm squares. Alternatively, standard 22-mm square
cover slips (Coming, Anton, NY, sizeno. 1) can be scribed with a diamond point
pencil. We scribe a cross over a 2- to 4-mm circle, yielding four separatequadrants
that can beused for different injection mixtures. The orientation of the four quadrants
is establishedby scribing one of the lines of the cross with an extension to the left
(similar in shapeto the number 7). The averagequadrant will contain 20-100 cells,
depending upon cell type anddensity.After scribmg, cover slipsare soakedfor 1h in
ethanol and dry-sterilized at 180C. Alternatively, the scribed slips can be removed
from ethanol, carefully flamed, and placed in a sterile culture &sh prior to plating
cells. Cells platedon coverslips aregrown in a humidified 5% CO, incubator until
ready for microinjection. Cells should not be removed from the incubator for microinjection for more than 15min at atime in order to minimize the rise in pH that occurs
in carbonate-basedDMEM.Altematively,during microinjection,cellscanbe maintained
m carbonate-free DMEM buffered with 10 mM HEPES, pH 7.4,
Cell Microinjection
177
178
3. Methods
3.1. Microinjection
Methods and Instrumentation
Mammalian-cell micromjection is best performed under phase microscopy.
The tip of the microcapillary loaded with the microinjection sample 1s posltioned above the target cell by the use of a micromampulator and an appropnate volume is transferred into the cytoplasm or nucleus by air pressure supplied
by a syringe or by an automatic injection device. Three types of micromanipulator setups can be used for cell microinjection. The first is the oil-type hydrau-
Cell Microinjection
779
180
be injected on the stage of the microscope and focus the objective on the plane
of the cells. The coarse controls consist of three knobs that direct movement in
the x-axis (left and right), y-axis (front and back), and z-axis (up and down).
Use the knobs on the coarse movement manipulator to bring the microcapillary
to the center of the culture dish and to lower its tip to just below the surface of
the culture medium.
Looking through the microscope at a x 100 magnification, focus on the
microcapillary tip by moving the microcaplllary back and forth with the joystick in the Y direction to locate the microcapillary as a moving shadow, and
raising the objective with the focusing knob until the tip of the microcapillary
comes into view. Change the objective lens to give a final magnification of
x200 or x400. Focus on the tip of the microcapillary and check that it is intact.
In 3- to 4-step Increments, first lower the focal plane with the focusing knob
and then lower the microcapillary tip with the course control until the tip is in
focus. Finally, when the target cells are in focus, bring the microcapillary tip
down, leaving itjust above thefocalplane of the cells, i.e., the tip is kept out of
focus to avoid penetrating the cells.
3.1.1.2. MICROINJECTION OF CELLS
Use the joystick assembly controls for fine movement of the microcapillary
and for microinjection. The joystick assembly of the Narashige micromampulator contains two knobs at the base which control fine movement separately m
the X- or y-axis. Deflection of the joystick causesthe microcapillary to move m
a combinedxy-axis. Rotation of the joystick raises or lowers the mlcrocapillary
in the z-axis. Use the two knob controls on the joystick assembly to center the
microcapillary tip over the area of cells to be microiqected. Rotate the joystick
to lower the tip to Just above the surface of the cell to be injected, the search
plane (the tip should appear slightly blurry m the search plane). Microinject
cells by carefully lowering the tip to penetrate the nucleus or the cytoplasm of
the cell as desired. The tip of the capillary that is positioned at a 45 angle will
penetrate the cell vertically, i.e., at a 90 angle. Successful injectlons are
indicated by gentle swelling of the nucleus or the cytoplasm, Move the
microcapillary tip from cell to cell by appropriate deflection of the joystlck.
When all of the desired cells in a field are injected, move to the new area by use
of the joystick knobs.
During cell microinjection, it will be necessary to periodlcally regulate the
pressure applied to the microcapillary by adjustment of the Gllmont syringe.
Injections with the manual system are performed under constant pressure,
resulting in a constant flow of fluid. Therefore, injections into the nucleus will
also deliver some sample into the cytoplasm. By constant pressure and by
operator control of the degree of nuclear or cytoplasmic swelling, a moderate
Cell Microinjection
181
182
Green, Thorh.m,
and Loewenstein
Usmg the micromanipulator keypad (M pad), switch to the Fast mode with
the SPEED toggle key and to Axial movement with the AXIAL key. The M
pad dtsplay should now read Dynamic, Fast, AX (axial movement), In (iqect
mode), X, Y, and 2 coordmates m microns (0 when the mstrument is first
turned on), and JB = INJ/IMP (Joystick button m the mJect/impale mode). If
the above parameters are not displayed, enter the correct ones using the M pad
keys. Center a 35-mm culture dish containing the cover slip with cells to be
injected above the obJective on the microscope stage and focus on the target
cells using x 100 magnification. Visually bring the tip of the microcaptllary
slightly past the center of the mtcroscope obJective by rotating the Joystick
knob clockwtse to lower the microcapillary, m the stmultaneous X/Z dnection
(i.e., a 45 angle smcethe AXIAL movement key IS toggled on).
After centering the microcapillary tip, toggle off the AXIAL movement key
so that the microcaptllary is now lowered in the Z rather than the X/Z direction.
Carefully lower the microcapillary by clockwise rotation of the joystick until
the tip isJust below the surface of the culture medium Switch to Slow using
the SPEED toggle key on the M pad to permit a controlled approach to the
plane of the cells, While viewing through the microscope, raise the obJective
wtth the focusmg knob to focus on the tip of the microcapillary by moving the
joystick back and forth m the Y direction to locate the mtcrocapillary as a
moving shadow, and ratsing the objective until the tip comes mto view.
Change the ObJective to provide a final magnification of x200 or x400. Focus
on the microcapillary tip and check that it is intact. Press the CLEAN key on
the T pad and confirm that the mJection mixture flows from the tip, as indtcated by the schlieren pattern (the clean function flushes the microcapillary
Cell Microinjection
783
at maximum pressure of approx 7000 hPa). In three to four steps, alternately lower the focal plane with the focus knob and then lower the
microcapillary with the joystick until its tip is in focus. At the last step, when
the target cells are in focus, carefully lower the microcapillary tip until it is
slightly above the focal plane of the cells, i.e., keep the tip slightly out offocus
to avoidpenetrating the cells.
3.1.2.4.
Check the injection parameters on the A4 pad by pressing the SET key and
then the INJECT key. Use the SET key to toggle between the choices and use
the UP or DOWN key as a toggle to change the choices. Ensure that INJ is on,
IMP is off, and for most injections, the Axial-injection mode is on (see Note
1). The speed of injection can be modified (300 pm/s is a good choice). Finally,
press the ENTER key to exit the program mode. On the T pad display, N indicates the total number of injections attempted; set N to zero prior to each new
series of injections.
The Z-LIMIT is the final depth of the microcapillary tip upon injectlon of
adherent cells and is experimentally established for each series of InJections.
The Z-LIMIT must be set before microinjection can be performed. Carefully
rotate the joystick clockwise to lower the microcapillary tip until it just touches
the surface of the target cell. Set this temporary Z-LIMIT into memory by
pressing SET, LIMIT, and ENTER on the Mpad. Raise the microcapillary tip
to the SEARCH PLANE with the joystick so that it is slightly above the surface of the cells. The image of the tip in the SEARCH PLANE will appear
slightly out of focus. Attempt to inject a cell by positioning the tip slightly
beyond the site of the intended injection and pressing the center button on the
joystick to automatically inject the cell. The microcapillary tip will be withdrawn in the X direction, will pierce the cell in the Axial direction down to the
Z-LIMIT, and will then be withdrawn to its original position in the SEARCH
PLANE. If injection is successful, as indicated by a gentle swelling of the targeted nucleus or cytoplasm, the current Z-LIMIT is satisfactory. Most often,
setting the Z-LIMIT will require several adjustments. If the tip passesthrough
the cell entirely, as indicated by a white spot that persists, adjust the Z-LIMIT
upward. If the tip does not puncture the cell, adjust the Z-LIMIT downward.
To adjust the Z-LIMIT, press the LIMIT key, the UP or DOWN key (each
press of the key adjusts the Z-LIMIT 160 nm), and then ENTER.
3.1.2.5. SEMIAUTOMATIC INJECTION OF ADHERENT CELLS ON COVER SLIPS
Cells are injected by positioning the microcapillary tip, while in the
SEARCH PLANE, over the target site and pressing the button on the joystick.
184
To inject the nucleus, position the tip of the capillary slightly beyond the center
of the nucleus. To inject the cytoplasm, position the tip over the cytoplasm
near the outer edge of the nucleus. Once an appropriate Z-LIMIT is set, cells in
the same focal plane are readily injected without modification of the Z-LIMIT.
However, uneven surfaces on the cover slip or culture dish or differences in
cell morphology may require periodic readjustment of the Z-LIMIT.
Depending on the sample inJected, the characteristics of the microcapillary
tip, and the condition and type of target cells, it may be necessary to adjust the
injection pressure (Pi) and injection time (Ti) to obtain gentle swelling of
the target cell. Adjustment is accomplished, by directly accessing the appropriate key on the T pad and adjusting to the desired value with the UP and DOWN
keys, followed by the INJECT MODE key. If debris adheres to the tip, it can
often be removed by briefly pressing the CLEAN/HOME key on the M pad,
which will withdraw the tip from the medium. Pressing of the CLEAN/HOME
key again will return the microcapillary to its original position. If the tip
becomes plugged, raise it slightly above the SEARCH PLANE by rotating the
joystick counterclockwise and focus on the tip. Then press the CLEAN key on
the T pad to expel liquid at 7000 hPa which will unplug the tip, as visualized by
the schlieren pattern under the microscope. After unplugging the tip, return to
the SEARCH PLANE and continue cell injections. If the plug cannot be
expelled, load and position a new microcapillary and continue microinjection.
When changing the culture dish and/or the microcapillary, press and hold
down the CLEAN/HOME key on the M pad. This action will withdraw the
microcapillary to the limits of the X and Y motors. When changing the
microcapillary, close the transjector valve by selecting valve position X with
the T pad. After changing a femtotip or a manually drawn capillary (no longer
than the previously used capillary), press the CLEAN/HOME key again to
return to within 700 microns of the original SEARCH PLANE. Then re-establish both the Z-LIMIT and the SEARCH PLANE. For a manually drawn microcapillary that is longer than the previous mtcrocapillary, cancel the home function
by pressing the CANCEL key. Then position the microcapillary above the cells
and establish the Z-LIMIT and SEARCH PLANE as described above.
Cell M/croinjection
185
permeablllzed, and incubated with the primary antibody that recognizes the
protein of Interest, and then stained with a second fluorochrome-conjugated
antibody that recognizes the primary antibody. Optimal fixation conditions
for each antigen-antibody interaction must meet two main criteria: the cell
structure should be preserved, and the antigenic structure should be maintamed in a form that allows recognition by the antibody. The optimal
fixative is determined empirically. There are two mam classesof tixatlvesorganic solvents such as alcohols or acetone and crosslinkers such as formaldehyde or glutaraldehyde. Some commonly used organic fixatives are 100%
methanol, 50% methanol/50% acetone, and 5% acetic acid/95% ethanol. Commonly used crosslinkers are 3.7% formaldehyde in PBS and 4.0% paraformaldehyde in PBS.
It 1soften necessary to block nonspecific interactions to prevent high fluorescence backgrounds. This is often achieved by incubating permeabilized cells
with PBS containing 5% BSA for 30 min. Using PBS/O.l% Tween-20 in the
blocking, washing, and incubation solutions will also help to maintain a high
signal-to-noise ratlo. Below 1sa general protocol that has been found useful for
many antigens.
At the completion of the micromjectlon experiment, transfer cover shps (cellside up) to new 35-mm2 dishes, rinse twice with PBS, and fix cells by incubation
with 2 mL of 3 7% paraformaldehyde/PBS for 10 mm. Aspirate the solution and
permeablhze cells by mcubatlon with 0.3% Triton X-lOO/PBS for 3 mm. Rinse
cover slips twice with PBS and store m PBS at 4C until ready for staining
Rinse cells on cover shps twice with PBS/O 1% Tween-20 and block cells by
incubation in PBS/S% BSA for 30 mm. Carefully plpet 50 pL of an appropriate
dilution of primary antlbody m PBS/O 1% Tween-20 onto the central area of
Injected cells (prior to use, centrifuge diluted antibody for 10 mm at 14,OOOg
to remove aggregates) Incubate cover slips m a humidified chamber for 60 mm
at 37OC
Washcells three times for 5 min with 3 mL of PBS/O.1% Tween-20 on a laboratory rocker.
Block cells with PBS/S% goat serum for 30 mm
Add to the cover shp 50 pL of an appropnate dilution of the secondary antibody m
PBS/O 1% Tween-20, e.g , l-100 dilution of goat antirabblt IgG coqugated with
either FITC or Texas red Incubate m a humidified chamber for 60 mm at 37C.
Wash cover slips three times with PBS/O. 1% Tween-20.
Using a fine forcep, dip each cover slip m water for 10 s, dram briefly against a
Kimwlpe, and place cell-side down on a drop (approx 25 pL) of mountmg
medium (see Note 2) placed on a microscope slide. Gently press the cover s11p
down (a pencil eraser works well) and remove excess fluid with a Klmwlpe Seal
edges of the cover slip with clear nail polish for permanent storage The cells are
now ready for fluorescence microscopy and photography (see Note 3).
786
187
Cell Microiqection
3 Incubate cover sltps for 30 min m 1 5 N HCl to denature cellular DNA Wash
coverslips three times for 3 mm with PBS.
4 Incubate cover slips for 30 min at 37C in a humidified incubator wtth 50 ml of
primary antibody solution consisting of a 1:50 dilutton m PBS/O.S% BSA of antiBrdU monoclonal antibody (Becton Dickmson, Rutherford, NJ, cat no 7580)
5. Wash cover slips three times for 5 mm with PBS.
6 Incubate cover slips for 30 mm at 37C with a secondary-antibody solution conststmg of 50 p.L of a 1.50 dilution in PBS/O.S% BSA of Texas red ConJugated
sheep antimouse irrununoglobulin polyclonal antrbody (Amersham, Arlmgton
Heights, IL, cat no N 203 1).
7 Wash cover slips three times with PBS, rinse briefly with water, and mount each
cover slip cell-stde down on a mtcroscope slide (see Note 2)
of microinJected
RNA
188
3 2.4
3.2 4.2
PREHYBRIDIZATION
1. Remove each cover slip from 70% ethanol with a fine-tipped forceps, blot the
edge agamst a KImwipe, place carefully m a stammg dish contammg 10 mL of
5 mA4MgC12/PBS, and Incubate for 10 mm. Be sure to monitor which side of the
cover slip contains the cells (mark one end of the staining dish and place cover
slips with cells facing the marked end) (a VWR Scientific staining dish, cat no
25452-002, contains four pan-s of grooves and can accommodate seven cover
slips positioned m a zigzag onentatlon).
2. For antisense RNA probes, acetylate cells by mcubatlon for 10 mm with 10 mL
of 0.1 M triethanolamine-HCl,
pH 8.0 containing 0 25% acetic anhydride (stock
0 1 M triethanolamme 1s brought to pH 8.0 with approx 2 mL of concentrated
HCI and stored at room temperature). Add 25 pL anhydrous acetlc anhydride to
10 mL of triethanolamine just before use Rinse cover shps twice with 2X SSC
(SSC IS 150 mMNaCl/lS
tiNa
Citrate, pH 7 0)
3 Aspirate the solution and incubate cover slips for 10 mm m the staining dish with
10 mL of 0.1 M glycme/0.2 M Tns-HCl, pH 7.4,
4. Aspirate the solution completely and incubate cover slips for exactly 10 mm
in 10 mL of 50% deionized formamide/2X SSC/lO mMDTT at 65C (do this
at the same time as the probe 1s being denatured, Just prior to hybndlzatlon).
Molecular-biology
grade formamide is deionized by stirring for 30 min with
AG5OlX8(D) resin (Bio-Rad, Hercules, CA), filtered through Whatman no. 1
filter paper, and then stored in the dark at -20C.
3.2 4.3.
HYBRIDIZATION
1. Dry down the following mixture m a separate Eppendorf tube for each cover slip
(Savant, Hlcksvllle, NY, Speed Vat Concentrator or a lyophllizer) Add in order
to each tube: 2 pL of 10 mg/mL (10 pg) E colz tRNA (Boehrmger); 2 pL of
10 mg/mL (10 pg) salmon-sperm DNA (Sigma, sheared to approx 300 bp by
passing through a 26-gage needle); and 620 ng of either 35S-labeled DNA or
35S-labeled RNA probe (l-5 x lo6 cpm).
2 Add 10 & of formamide to the dried probe mixture m each Eppendorf tube.
3 Cap each Eppendorf tube and denature the probe by heating for 10 mm at 90C
using a heating block
Cell Microinjection
4. Prepare 2X hybridization buffer by mixing l/5 volume of 20X SSC, l/5 of BSA
(20 mg/mL, Boehrmger, molecular-biology grade), 215 volume of 50% dextran
sulfate (Pharmacra), and l/5 volume of 1 M DTT (dithlothreitol). One at a time,
remove an Eppendorf tube from the heating block, add 10 pL of 2X hybrrdization
buffer, and gently pipet up and down five times with a 20-pL prpetman (the final
concentration in the hybridization mixture is 2X SSC or 0.36 MNa+). Take every
precaution to avoid an bubbles. If air bubbles are formed, centrtfuge the tube for
10 s Transfer the hybridization mixture in two installments (to avoid an bubbles)
to a sheet of parafilm taped tightly on top of a glass plate. With a pair of fine
forceps, remove the cover slip from the prehybridization mixture, drain briefly,
and place it cell-side down on top of the 20-$ drop of hybridization mixture.
Remove any an bubbles over the region of injected cells by gentle manipulation
of the cover slip with the forceps. When all cover slips have been applied to the
probe mixture, place a second sheet of parafilm over the cover shps to prevent
drying, and incubate at 37C for DNA probes or 45C for RNA probes in a
humidified incubator for 4 h to overnight.
3.2.4.4.
1 Gently lift each cover slip from the parafilm with a pair of forceps as follows. To
avoid breaking fragile cover slips, add a drop of 50% formamide/2X SSC to the
edge of the cover slip, pierce the parafilm near the edge wrth sharp forceps, and
gently break the surface tension by slowly raising the cover slip to allow the
solution to enter the space between the cover slip and the parafilm. Incubate the
cover slips m 10 mL of 50% formamide/2X SSC for 30 mm at 37C in a staining
dish (seven slips per dish as described above).
2 Aspirate the solution and incubate the cover slips for 30 mm in 10 mL of 50%
formamide/lX
SSC at 37C.
3. Transfer each cover shp to individual 60-mm culture dishes contammg 10 mL of
1X SSC and wash by rocking for 30 mm. Repeat washes two or three times.
4. Dehydrate the cells on cover slips in a staining dish by sequential incubation for
5 mm with 70,95, and 100% ethanol. Air-dry for at least 30 min.
5. Mount each cover slip cell-side up at one end of a microscope slide using a drop
of Pro-Texx mounting medium (American Scientific Products [McGraw Park,
IL] cat no. M7635). Mounted cover slips are ready for autoradiography after
approx 1 h.
3.2.4.5.
1. Gently lift each cover slip from the parafilm as described above and wash m
individual 60-mm culture dishes by rocking for 60 min m 10 mL of 4X SSC/
10 mA4DTT
2. Transfer cover slips to a staining dish. Dehydrate by 2-min sequential incubations in 70% ethanol and 95% ethanol containing 300 mA4 ammomum acetate,
followed
by 100% ethanol.
190
3.2.4.6. AUTORADIOGRAPHY
1 In a darkroom under a safelight (Kodak Wratten no 2 safehght contammg a
25-W bulb), melt a 20-mL aliquot of Kodak NTB-2 emulston in a 50-mL
centrifuge tube in a 42C water bath for at least 1 h (Kodak NTB-2 emulsion
on receipt from the vender is melted at 42C m the dark for at least 2 h and
20-mL ahquots are placed m plastic tubes, wrapped in alummum foil, and
stored at 4C until used).
2. Carefully and slowly dip the cover slip end of the slide into the emulsion. Allow
the mounted cover shp to dram vertically in a test tube rack for approx 30 mm in
the absolute dark (as the emulsion dnes, it becomes very light sensitive, even to
a safelight).
3. Place shdes in a light-tight slide box contammg a desiccant, wrap the box m
aluminum foil, and expose the emulsion-covered cover slips at 4C for 2-4 d
depending on the strength of the radioactive signal
4 After exposure, allow slides to warm to room temperature.
5 In a darkroom (no safelight), develop the emulsion-covered cover slips mounted
on slides m a shde-staming tray for 5 mm at approx 18C m Kodak D- 19 developer diluted 1: 1 with water
6. Rinse slides for 30 s m water at approx 18C
7. Fix slides for 5 min in Kodak fixer at approx 18C
8 Wash slides for 30 mm m cold runnmg water Air-dry slides
9 Score slides by microscoptc observatton under phase at a magmficatton of
x200-400 Heavily hybridtzed cells ~111have many exposed silver grams visible
over the cell Lightly hybrtdized cells ~111 show individual grains, often best
viewed out of phase, and can be quantitated by gram countmg See Note 6 for
combined IF and autoradiographic analysis.
Cell Micromjection
4. Notes
1. The Eppendorf micromanipulator provides the ability to automatically inject m
two different modes, the Axtal mlectton mode on and the Axial injection mode
off: In both modes, the capillary IS held at a 45 angle With the Axtal mode on,
mjection 1s automatically performed with the cell being pierced m the Axial
direction, i e., at a 45 angle, and then returned to its initial position at the Search
Plane. With the Axial mode osf, the capillary (which is normally held at a 45
angle) is lowered vertically m the X direction, thus injecting the cell and then is
returned to its initial position. The Axtal injectton mode on is the preferred
method for injecting most mammalian cells. However, the Axial mode off has
been found to improve survival of certain cell types, including human cardiac
myocytes and prostate cells
2 Several commercially available mounting media are available for permanently
attaching cover slips cell-side down to slides. For fluorescence microscopy, we
recommend a SlowFadeTM-light Antifade kit from Molecular Probes. A permanent mountmg medium useful for both fluorescence and X-Gal stammg 1s
Gelvatol, which can be used for fluorescent microscopy by the addition of
DABCO as described (ref. 17).
3 It is Important to photograph all important data for permanent records. For blackand-whtte photography, we use Kodak T-Max (ASA 400) with a microscopic
magnification of x20&400.
4 High levels of P-galactosidase expression produce detectable blue cells within
60 min after addmon of the X-Gal solution. For cells with less /3-galactosidase
activity, overnight stammg may be required and therefore stained cells should be
monitored by phase microscopy to determine when to terminate X-Gal staining.
Note that when coinjectmg a fluorescent marker, X-Gal staining which 1scaused
by a blue precipitate will quench the mununofluorescent signal from the marker.
Because P-galactosidase activity is derived from an injected plasmrd, any blue
stamed cells have been successfully injected whether a fluorescent marker is seen
or not. Thus the total number of injected cells is the sum of blue-stained cells plus
fluorescent cells not exhibiting a blue stain.
5. BudR can be added to cells for dtfferent time periods, depending on the purpose
of the experrment. For example, to measure quiescence or stimulation of quiescent cells by serum or microinjection of various oncogenes or mttogens, it is
convenient to add BudR at 4-8 h after micromjection followed by mcubation for
12-24 h prior to fixation and analysis for BudR incorporation. In this manner,
one can determine the cumulative number of cells that enter S phase during the
mcorporatron period. Pulse mcorporation of BudR for shorter periods at various
times after microinjection can be used to more precisely define the time periods
when cellular DNA synthesis is occurrmg.
6. It is possible to detect both protein by immunofluorescence and RNA by in sztu
hybridization. First, do standard zn sttu hybridization analysis but do not add
photographic emulsion Second, perfomr the antibody-staining reaction Third,
coat the slide wtth photographic emulsron, expose, and develop. Under the
192
microscope, one can now observe the same field by fluorescence microscopy for
antibody-reacting cells and by phase microscopy for radIoactIve grams over the
hybridization positive cells.
Acknowledgments
We thank C. E. Mulhall for editorial assistance and Rob Kern (Eppendorf
Scientific) for expert advice and mstruction on the use of the Eppendorf
Mlcromjectlon System. The experiments on cell mlcroqectlon reported here
were supported by Public Health Service grants CA-2956 1, CA-54703, AI-2820 1,
and Research Career Award AI-04739 to M G from the National Institutes
of Health.
References
1 Capeccl, M (1980) High efficiency transformation by direct mlcromJectlon of
DNA mto cultured mammalian cells Cell 22,479-488
2 Shenk, T. (1996) Adenovlridae: the viruses and their repllcatlon, in Vwology
(Fields, B. et al,, eds.), Llppincott-Raven, New York, pp. 211 l-2148.
3. L&e, J. W , Loewenstein, P. M., Green, M. R., and Green, M. (1987) An adenovu-us E 1A protein region required for transformation and transcriptional repression Cell 46, 1043-l 05 1.
4 Green, M., Loewenstem, P M., Pusztal, R., and Symmgton, J. S (1988) An adenovirus EIA protein domain activates transcription m vivo and m vitro m the
absence of protein synthesis Cell 53,92 l-926.
5 Song, C -Z., Tlerney, C J , Loewenstein, P. M , Pusztal, R., Symmgton, J S ,
Tang, Q -Q., Toth, K., Nlshkawa, A., Bayley, S T , and Green, M. (1995) Transcriptional repression by human adenovirus E 1A N-terminus/conserved domain 1
polypeptides m vivo and in vitro m the absence of protern synthesis. J Blol Chem
270,23,263-23,267.
6. Stabel, S , Argos, P , and Phihpson, L (1985) The release of growth arrest by
microinjection of adenovnus. EMBO J 4,2329-2336
7. Kaczmarek, L., Ferguson, B., Rosenberg, M., and Baserga, R. (1986) Induction of
cellular DNA synthesis by purified adenovirus ElA proteins. Vzrology 152, l-10.
8 Ruley, H. E. (1983) Adenovlrus early region IA enables viral and cellular
transforming genes to transform primary cells in culture. Nature (London)
304,602-606
9 Mulcahy, L. S , Smith, M R., and Stacey, D. W. (1985) Requirement for ras protooncogene function during serum-stimulated growth of NIH 3T3 cells. Nature
(London) 313,24 l-243
10. Dobrowolski, S , Harter, M., and Stacey, D. W (1994) Cellular ras activity 1s
required for passage through multiple points of the GdG, phase in BALB/c 3T3
cells. Mol. Cell. Blol. 14, 5441-5449
11 Riabowal, K. T., Vosatka, R. J., Ziff, E. B., Lamb, N J., and Feramlsco, J R.
(1988) Micromjectlon of fos-specific antibodies blocks DNA synthesis m fibroblast cells. Mol Cell Bzol 8, 1670-1676
Cell Microinjection
193
12. Kovary, K. and Bravo, R (1992) TheJun and fos protein families are both requtred
for cell cycle progressron in fibroblasts. Mol Cell. Biol 11,4466-4472.
13. Arias , J., Alberts, A S , Brindle, P , Claret, F. X , Smeal, T , Karm, M.,
Feramisco, J., and Montminy, M. (1994) Activation of CAMP and mrtogen
responsive genes relies on a common nuclear factor Nature (London) 370,
226-229.
16
Immunoprecipitation
of El A-Containing Protein Complexes
Elizabeth Moran and Peter Yaciuk
1. Introduction
Important insights into cell growth and transcriptional regulation have been
realized by correlating adenovirus E 1A-induced cell-growth-control mechanisms with El As specific interactions with host-cell proteins (see ref. 4). A
key method for directly demonstrating these El A-host cell protem tnteractions is by immunoprecipitation. This assay is performed under condttions that
efficiently lyse cells, but preserve protem-protein interactions. This method
combines the elegant specificities of monoclonal antibodies and the fortuitous
properties of nonionic detergents. These highly efficient and specific means of
isolatmg protein complexes from cell lysates, while detailed here for ElAcontaining protein complexes, have general application for the study of other
cellular-protein complexes.
When investigating potential protein-protein interactions of a specific protein, it is important that the investigator optimize assay conditions that both
mirumize the amounts of nonspecific-interacting proteins and maximize the
amount of specific-interacting protein. Procedures to minimize the amounts of
nonspecific-mteractmg protein are detailed below. These steps, along with
immunoprecipitations done with control antibodies, should help to identify
candidate proteins that specifically interact with the protein of interest. Once a
candidate specific-interacting protein (or proteins) is (or are) identified, the
investigator needs to demonstrate that the candidate specific-interacting protem is coimmunoprecipitated through protein-protein interaction and not
through a direct recognition by the antibody used. To address this issue one
should demonstrate that the antibody used to immunoprecipttate the protein
complex will not recognize the candidate specific-interacting protein by WestFrom
195
and Protocols
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796
2. Materials
2.1. Radiolabeling
of Cultured
Cells
1 Tissue-culture medium, tissue culture cells, tissue culture plates (see Note 1).
2 Mammalian cells that constuutively express El A-gene products, such as 293 cells
(ATCC, Manassas, VA, CRL 1573), or other specific cell systems where El A
expression can be mtroduced (see Note 2).
3 Methlonme/cysteme-free tissue culture medium (Life Technologres, Garthersburg,
MD, cat. no. 21013-016).
4 3SS-labeled methionine and cysteme (Tran35slabel, ICN Bromedicals, High
Wycombe, UK, cat. no 5 1006 or ExpressTMethionine/Cysteine Protem Labelmg
MIX, NEN Life Sciences Products, Boston, MA, cat. no NEG072; see Note 3)
1. Lysrs buffer: 0.1 % nomdet P-40 (NP40) (see Note 4), 20 mM sodmm phosphate,
250 mI4 sodium chloride, 30 mM sodium pyrophosphate, 5 mA4 EDTA, 10 mM
sodium flourlde (see Note 5) Add drthrothreltol to 5 mA4 final concentration
from 1 M dithiothrertol stock (see Note 6).
2 Protease-inhibitor
stock solutions aprotmm (5 mg/mL, add 1 pL/mL lys~s
buffer), leupeptin (5 mg/mL, add 1 pL/5 mL lysis buffer), pepstatm (5 mg/mL,
add 1 @J5 mL lysis buffer), phenylmethylsulfonyl
fluoride (PMSF; see Note 7)
(75 mg/mL m DMSO, add 5 p.L/mL lysis buffer) or use commercially avarlable
E lA-Containing
3.
4.
5.
6.
7.
Protein Immunoprecipitation
197
2.3. Norma/izafion
of Cell Lysafes
1. Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, cat. no. 500-0001, or other
venders, see Note 14).
2 Glass-microfiber filter disks (Fischer Sctenttfic, Ptttsburgh, PA) (Whatman GF/C,
cat. no. 1822-024 or various vendors).
3 20% trichloroacetic acid
3. Methods
3.1. Radiolabeling
of Cultured
Cells
3.2. Immunoprecipifafion
1 After the mcubatton period, remove radioacttve media into designated radioactive waste containers.
2. Add 1 mL lysis buffer to each plate. Distribute lysis buffer over entire surface of
plate, manually shake, and then rock plates m cold room for 10 mm
198
assay conditions.
1. Distribute 800 pL dHzO to a series of SIX mtcrofuge tubes for standards and
duplicate tubes for each cell-lysate sample.
2. Add 5 pL of lysis buffer to the SIX protein-standard tubes
3. Add 0,2,5, 10, 15, and 20 g of the IgG-protein standard to the SIX protem-standard tubes, respectively.
4. Add 5 & of cell Iysate of each sample to the corresponding sample tubes
5 Add 200 & of the dye-reagent concentrate to each tube and mix well. Incubate at
E IA-Containing
Protein lmmunoprecipitation
199
4. Notes
1 A wide variety of tissue-culture medium and serum concentrations and types are
used for growing specific cell types in culture For 293 cells, grow m 10% calf
serum m DMEM contaming streptomycin and penicillm
2. The E IA protems have been introduced into numerous types of cells by transfection, such as the primary human-embryonal kidney-cell line called 293 cells, m
which sheared human adenovirus DNA was transfected and a transformed cell
lme that constitutlvely expresses the El A gene products was isolated (see ref. 2)
Since many cell types are permissrve for adenovirus mfection, the E 1A products (and its cell growth control mechanisms) can be introduced into a wide
variety of cells to address specific scientific questions about particular biologtcal mechanisms.
3. All labeling and handling of radioactive materials should be done m comphance
wtth Nuclear Regulatory Cormmssion Radiation Safety Regulations. Tran3?Slabel and ExpressTMMethionme/Cysteine
Protein Labelmg MIX are cellular
hydrosylates of E colz grown in the presence of 35S04. Typically, they contain
270% L-methionine, 5 15% L- cysteine, plus other label compounds, but serve as
an economical source of 35S-labeled amino acids and are good for general m vivo
35S-labeling of cellular proteins Please note that 35S- labeled compound have
volatile components that can be inhaled or contaminate the working areas. It IS
Important to imttally open the 35S-reagents stock vial in a fume hood and check
for and clean up any working surfaces, mcludmg CO* mcubator shelves! It IS a
good idea to equip tissue-culture incubators that circulate the Incubator an
through a sterilization filter with a charcoal prefilter to remove any volatile 35Scompound. To maximize the m vivo mcorporation of these radioactive ammo
acids it is best to use them in conjunction with methionine/cysteine-free DMEM
Increases in incorporation have been noted by preincubating cells for up to 1 h m
methionme/cysteme-free
DMEM to deplete cellular methtonine/cysteine con-
200
6.
7.
8.
9,
10.
11,
HA-Containing
Protein Immunoprecipitation
201
12 Whereas antibodies are rather stable protems, the buffers are ideal media for
growth of contaminating microbes Antibody solutions should be filter-sterthzed
and treated with strict aseptic technique. Store each antibody solutton in multtple
portions to prevent loss of the entire preparation by a single contammatton event.
It IS recommended that multiple portions of antibody solution be frozen, while
keeping one portion at 4C as the current working stock. This will avoid loss of
antibody affinity because of the detrimental effect of multiple freeze/thaw
cycles and indefimtely preserve the mittal preparation. Addition of bacterttidal agents, such as sodmm azide (at 0.02% (w/v)) or mercury-[(o-carboxyphenyl)thto]-ethyl
sodium salt (at 0.01% w/v) (thtmersol, Sigma, T-5125)
work well m preventmg mtcrobial contammation in antibody soluttons wtthout affecting most antibody-antigen interactions, but it should be strongly noted
that these bactericidal agents are htghly reactive molecules that can affect antlbody affinity, m vttro btochemlcal reactions and are htghly toxic in viva Also,
antibody preparations should be titered to assure that the investigator IS in anttbody excess and that the amount of protein A Sepharose CL-4B beads is sufticient to bind that amount of antibody.
13. Protein A has a range of affinities for different types of antibodies. If protein A
does not recognize or has low affinity for your specific antibody, you can add a
secondary antibody to improve tsolatton of your protem of interest. For example,
some mouse monoclonal antibodies of subclass IgG, are not recognized by
protein A. In this case, by adding a titered amount of a rabbit antimouse secondary antibody, the IgGl IS recogntzed well by the secondary antibody and the
secondary antibody is recognized well by protein A, and your protein of Interest
can be isolated.
14 Sepharose protein A CL-4B beads (Pharmacia Biotech, cat no 17-0780-o 1) are
commerctally supplied and are freeze-dried m the presence of additives. When
the beads are rehydrated, they must be washed well to remove the additives It is
also important to vortex these beads thoroughly to disperse any clumps of beads.
These clumps have clogged pipetman tips without preventing buffer from bemg
drawn up mto the ptpet tip, resulting m the correct volume of solution transferred
but wtth reduced or no bead transfer and therefore reduced nnmunocomplex isolation To eliminate this problem, cut off the end of the ptpet tip (approx 2 mm)
with a clean scissor or razor blade. This step has led to more consistent final bead
volumes and mnnunocomplex isolation. Also, these protein A Sepharose bead
preparations will start to settle wtthm 1 mm if left standing. It IS critical to keep
slurry well mixed during transfer to multiple sample immunoprectpitation
tubes
to maintam consistent bead transfer.
15 Commercially available protein assay kits each have their advantages and dtsadvantages depending on the specttic contents of the protem-solutton buffers and
amounts or type of proteins being measured. To minimize the effects caused by
buffer contents, it 1srecommended that you add a volume of lysis buffer to your
protein standards that equals the cell-lysate volume that you use to do your protein measurements.
202
References
1 Bonifacino, J S (1987) Biosynthetic labeling of protems, m Current Protocols In
Molecular Bzology, Wiley, New York, Unit 10.18
2. Graham, F L., Smiley, J., Russell, W , and Nairn, R (1977) Characteristics of a
human cell lure transformed by DNA from human adenovu-us type 5 J Gen
Vlrol 36, 59-72.
3 Harlow, E. and Lane, D (1988) Antzbodres A Laboratory Manual Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY, pp 636-640.
4 Moran, E. (1994) Cell growth control mechamsms reflected through protein interactions with the adenovirus E 1A gene products, Semm. Vlrol. 5,327-340.
5 Neugebauer, J. M (1990) Detergents. an overview, m Guide to Protern Purljkation (Deutscher, M. P., ed.), Methods Enzymol. 182,239-253
Preparation of Splicing-Competent
from Adenovirus-Infected
Cells
Oliver Miihlemann
Nuclear Extracts
1. Introduction
Adenovn-us has contributed srgnificantly to our current understanding of the
orgamzatron and expression of genes in eukaryotic cells. The most startling
discovery was probably the observation that most adenovrrus mRNAs are
encoded from drscontmuous DNA segments. The splat-gene and RNA-sphcmg
concepts were rapidly extended to other systemsand accepted as dogma within
a few months after then initial discovery. It was clear right from the beginning
that most adenovirus-transcription units encode multiple alternatively spliced
mRNAs: Many are translated into proteins that have unique biologrcal activrties. It was also shown that the accumulatron of alternatively spliced mRNA
was temporally regulated during virus mfection. The shift from the early to late
pattern of mRNA accumulation was shown to be dependent on viral late-protein synthesis (1).
Much of our knowledge about the btochemrstry of RNA splrcmg comes from
studies of the adenovnus major-late-first intron. The conclusions from these
and other studies have been extensively revtewed elsewhere (2) and will
not be summarized here. Experiments to reproduce RNA splicing m vttro mltially progressed slowly. The first examples of successful m vrtro RNA splicing
were published in the early 80s (3-7). Success was to a large extent hampered by the difficulty of synthesizing or purifying a substrate RNA for in
vitro splicing experiments. A major step forward was therefore the development of the SP6 in vitro transcription system for pre-mRNA substrate synthesrs (8). With an easy method to produce large quantitres of substrate RNA the
basic mechanisms of RNA sphcmg were rapidly established (2).
From
203
and Protocols
NJ
204
205
transcnptlon, but produces nuclear extracts that also are competent for m vitro
RNA splicing. However, we (and most other groups) extract nuclei with 0.6 M
KC1 instead of 0.42 MNaCl as described in the original protocol. The usage of a
higher salt concentration is essential to obtain nuclear extracts that reproducibly
sphce the regulated adenovlrus Ll-IIIa mRNA in vitro (10,11). Interestingly,
higher salt appears to extract a dtstinct factor required for adenovirus Ll-IIIa
splicing that does not have a major effect on splicing of prototypical transcripts
conforming to the RNA 3 splice site consensussequence (11). Substrate RNA 1s
synthesizedusing bacteriophage T7 or SP6 RNA polymerase. The efficiency of
in vitro splicing of pre-mRNAs with weak 3 splice sites, such as Ll-IIIa, is
sometimes low and therefore difficult to detect. Thus, we often include a Ul
snRNA binding site downstream of the 3 splice site to improve the efficiency of
weak splice-site usage (12). Such a Ul -tag can increase the efficiency of splicing
of weak splice sites up to 20 times. However, the inclusion of a Ul -tag may for
some experiments be counterproductive, because it results in an unproportlonal
increase of the splicing efficiency in uninfected nuclear extract (0. M., unpublished observation), resulting in an underestimation of the actual difference m
splicing efficiencies between infected and uninfected nuclear extracts.
2. Materials
2.1. Infection of HeLa Spinner Cells with Adenovirus
The protocol below 1sadapted for 4-6 L exponentially growing HeLa spmner cells, giving approx 10 mL nuclear extract, but can be eastly adjusted for
cell cultures ranging between 500 mL and 30 L. To prepare small-scale nuclear
extracts, see Lee et al. (13)
1. MEM
spinner-cell
medium
(can be obtained
Galthersburg,MD).
2 Newborn calf serum (Life Technologies).
3. Peniclllm (10,000 IU/mL)/Streptomycin
(10,000 pg/mL) stock solution (Life
Technologies),
4 Purified adenovirus stock (wild-type or mutant of your choice) with a titer >lOO
PFU per mL.
Nuclear Extract
SlOO Extract
3. Stock solutions for buffer preparation. Make up with autoclaved ddH,O 0.5 A4
HEPES pH 7.9 (Sigma, St Louis, MO, H 0891), adjusted to pH 7.9 with 5 A4
206
4.
7.
8.
2.3. Preparation
of Radioactively
Labeled Splicing
Substrates
207
3. Methods
3.1. Infection
208
3.2.1. Nuclear-Extract
Preparation
1. Check the cell dens@ at the end of mfectton. It should be almost the same as at
the start of mfectton An adenovn-us infection mhrbrts cell drvtsron Thus, rf cells
continue to divide during the course of mfectron, the mfectton most probably drd
not work
2 Collect cells by centrtfugation at 18OOg at 15C for 20 mm (Beckman J6M/E
centrifuge, JS-4 2 rotor.) Decant medium (see Note 2) and resuspend cells gently
m a small volume of remaining medmm. Transfer mto two, conical 250-mL centrifuge bottles
3. Pellet cells at 1OOOgat 4C for 10 mm (Beckman GPKR centrifuge). Carefully
remove all medium and resuspend cells in 20 mL ice-cold PBS and pool mto one
bottle, measure volume. From here on all steps should be performed on ice (see
Note 4)
7
8
9.
10.
11.
12.
Determme the packed cell volume (pcv) according to the formula. total volume
of suspensron (step 3) - 20 mL = pcv.
F111up wrth ice-cold PBS and repeat centrrfugatron as m step 3
Decant PBS (carefully remove all remains of PBS with a Pasteur pipet) and
resuspend cells m (5 x pcv) mL ice-cold buffer A (hypotomc buffer) by gently
prpettmg up and down. Leave on ice for 10 mm
Transfer cells mto transparent 50-mL centrifuge tubes (note total volume) and
centrifuge at 500g at 4C for 10 min (Beckman JS 13.1 rotor)
Carefully remove the supernatant and measure Its volume. The volume of the
swollen cells is total volume (step 7) - supernatant and should by about
2-2.5 x pcv.
Add (2 x pcv) mL ice-cold buffer A and gently resuspend the cells with a prpet
Transfer the solution to a 40-mL homogenizer, prechilled on ice. Disrupt cells
with 10-12 strokes usmg the tight pestle To check the efficiency of disruption,
dilute a drop of the solution with buffer A, add 2 pL 0.4% trypan blue (Sigma),
and check under the microscope. Nuclei stain blue, intact cells do not stam. If
necessary, give a few additional strokes until approx 90% of the cells are disrupted (see Note 6).
Transfer the solution mto a transparent centrifuge tube (note volume) and spur at
750g at 4C for 10 min (Beckman JS 13 1 rotor)
Transfer the supernatant (cytoplasmtc fraction) into a new tube (note volume,
see Note 7). To prepare cytoplasmrc SlOO extract continue with this fraction
at step 19.
Splicing-Competent
Nuclear Extracts
209
13. Spm the pellet (the nuclet fraction) at 20,OOOg at 4C for 20 mm (Beckman JA20
rotor), then remove and discard the supernatant (note its volume) Calculate the
packed nuclei volume (pnv) accordmg to the formula. pnv = total volume after
cell dtsruption (step 11) - [cytoplasmic supernatant (step 12) + supernatant from
20,OOOg spin (step 13)]
14. Add (1 x pnv) mL of me-cold buffer C (high-salt buffer) to the nuclei pellet,
transfer to the small (IO-mL) homogenizer, and resuspend nuclei wtth several
(6-12) gentle strokes using the ttght-fittmg pestle.
15 Transfer the homogenate back to the centrifuge tube, embed it m ice, and shake
on a rocking table for 30 mm
16. Spin the homogenate at 20,OOOg at 4C for 30 min (Beckman JA20 rotor).
17 Collect the supernatant (nuclear extract) and dialyze tt agamst Ice-cold buffer D
at 4C usmg a membrane with a cut-off at 12-14 kDa Use 4 x 250 mL buffer D
and change buffer m 1-h intervals.
18. Spm the extract at 20,OOOg at 4C for 20 min (Beckman JA20 rotor), discard the
pellet, ahquot the supernatant mto Eppendorf tubes, quack-freeze m bqutd mtrogen, and store at -7OC (see Note 8)
simultaneously
with the
Substrates
210
on the efticiency of your Cerenkov counter. Calculate the total amount of dpm per
reactton according to the formula. dpm (total) = dpm (measured m ahquot) x 90
4 Add 1 uL of RQ DNase to the reaction and continue incubatron for 30 mm at 37C
5 Add 15 pL loading buffer to stop the reaction. Store samples at -20C or continue dnectly.
Splicing-Competent
Nuclear Extracts
211
step 2), transcrtpts up to 2 wk old will work; however, fresh transcripts usually gave a higher splrcing efficrency
Assay
Spm ahquots wtth desired amount of transcript immediately before use, wash the
pellet with 80% ethanol, remove all ethanol with an outdrawn, sterile Pasteur
plpet, and dry under the bench lamp for 3-5 min. Dissolve the pellet in autoclaved ddH,O to 20,000-25,000 dpm/$.
2. For a 25-pL standard reactron, mix the following Ingredients in a Eppendorf tube
on me. (see Notes 15-17)
5 pL 13% PVA.
5 pL Buffer D
10 pL Nuclear extract
1 0 & 62.5 n&I MgC& (see Note 14).
1 0 pL 0.5 M Creatme phosphate
0 5 pL 100 m&I ATP (see Note 14)
2.5 & RNA transcript, approx 50,000-70,000 dpm (5-10 fmol).
Collect maternal at the bottom of the tube by a short pulse m the mlcrofuge, mrx
by prpetting up and down, do not vortex, and Incubate for 90 mm at 30C
Add 175 pL protemase K mtx to each tube and incubate at 45C for 3&45 mm
(see Note 18)
8.
10
11.
4. Notes
I Cells are infected m a small volume of medmm (10 cells/ml) m order to achieve
efficient adherence of vu-us to cells and a somewhat synchronous infection. The
212
A
M-
f--m
r--J+TJ
A-
510
396
344
298
km
220
UmJ
154
11121
en
El
Fig. 2. (A) Time-course experiment showing the change in pre-mRNA, intermediates and product accumulation. In this experiment a penton-base minigene pre-mRNA
(16)was spliced in nuclear extracts prepared from uninfected HeLa cells. From a 160~&
splicing reaction, 25+L aliquots were removed after 0 (lane 2) 15 (lane 3) 30 (lane 4),
Splicing-Competent
3.
6.
7.
8.
Nuclear Extracts
273
45 (lane 5), 60 (lane 6), and 120 (lane 7) mm. RNA was isolated as described and
products resolved on an 6% polyacrylamide 8 A4 Urea gel Lane 1: Size marker. (B)
Comparison of the splicing efficiency of several major late mmrgene pre-mRNAs m
nuclear extracts prepared from uninfected (HeLa) and infected (Ad) HeLa cells (modlfied from ref. Id).
214
9, The DNA template for m vitro transcribed RNA can be a plasmld with an SP6
10
11
12
13
14,
15
Splicing-Competent
Nuclear Extracts
215
free exon 1 and decrease of the substrate RNA facilitates identification of these
bands The intermediates (lariat and lartat-exon 2) are branched molecules and
migrate at unpredictable positions m the gel and can easiest be Identified m a
time-course experiment by their accumulation. When duplicate samples are run
on two gels with different polyacrylamide concentrations, the intermediates are
the bands that change then posittons relattve to the size marker.
16 A reaction omitting ATP and creatine phosphate is a valuable negative control
17 For IZ reactions, always prepare master mixes for (n + 1) samples containing all
common ingredients to minimize pipetting errors. When PVA 1s included into a
master mix, viscosity of the solution changes and therefore approx 20% more
master mix than calculated should be prepared
18. For most transcrtpts, phenol/chloroform and chloroform extractions (Subheading 3.4., steps 4 and 5 in the protocol) can be skipped without any negative
effects on the result Precipitate RNA by adding 160 pL ddH,O, 40 @ 3 M
sodium acetate, and 1 mL 95% ethanol after the protemase K treatment (Subheading 3.4., step 3) However, note that some RNAs will give smeary bands
and a large proportion of radioactivity retarded m the well when these extractions
are omitted.
References
1 Imperiale, M , Akusjarvi, G , and Leppard, K (1995) Post-transcriptional control
of adenovtrus gene expression. Curr Topics Mcrobiol
199, 139-l 7 1.
2 Moore, M. Query, C C , and Sharp, P. A. (1993) Splicing of precursors to mRNAs
by the sphceosome, in the The RNA World (Gesteland, R F and Atkins, J F ,
eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 303-357.
Weingartner, B. and Keller, W. (198 1) Transcription and processing of adenovtral RNA by extracts from HeLa cells Proc Natl. Acad Scr USA 78,4092-4096
Kole, R. and Weissman, S M (1982) Accurate in vitro sphcing of human P-globin
RNA Nuclerc Acids Res 10, 5429-5445.
Hernandez, N. and Keller, W (1983) Splicmg of m vitro synthesized messenger
RNA precursors in HeLa cell extracts Cell 35,89-99.
Padgett, R. A., Hardy, S. F , and Sharp, P A. (1983) Splicing of adenovu-us RNA
m a cell- free transcription system Proc Nat1 Acad Scl USA 80, 5230-5234.
Kramer, A. R., Mamatis, T , Ruskm, B., and Green, M. R (1984) Normal and
mutant human /3-globm pre-mRNAs are faithfully and effictently spliced in vitro.
Cell 36,993-l 005
8 Green, M. R., Maniatis, T., and Melton, D. A. (1983) Human @globm pre-mRNA
synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell 32,68 1-694.
9 Dignam, J D., Lebovttz, R M., and Roeder, R G (1983) Accurate transcription
imtiation by RNA polymerase II in a soluble extract from isolated mammalian
nuclei. Nucleic Aczds Res 11, 1475-1489
10. Kreivi, J.-P., Zerivitz, K., and Akusjarvt, G (1991) Sequences involved in the
control of adenovtrus Ll alternative RNA splicmg. Nucleic Acids Res. 19,
2379-2386.
216
11. Zerrvttz, K., Krervt, J -P., and Akusjarvt, G (1992) Evidence for a HeLa cell
sphcing actrvrty that is necessary for activation of a regualted adenovn-us 3 splice
sate. Nucleic Acids Rex 20, 3955-3961.
12. Kreivt, J.-P., Zerivrtz, K., and AkusJarvi, G. (1991) A Ul snRNA bmding sate
improves the efticrency of m vrtro pre-mRNA sphcmg. Nucleic Aczds Res 19,6956
13 Lee, K., Zerivrtz, K., and Akusjarvt, G. (1995) Small-scale preparation of nuclear
extracts from mammalian cells, m CelZ Biology A Laboratory Handbook, vol 1
(Celis, J. E , ed.), Academic, London, pp. 668-673.
14. Eperon, I. C., and Kramer, A. R. (1993) Splicing of mRNA precursors m mammalian cells, m RNA Processzng, A Practml Approach, vol 1 (Higgins, S. J and
Hames, B D , eds.), IRL, Oxford, pp 757-101.
15. Ausubel, F. M., Brent, R., Kingston, R E , Moore, D D , Serdman, J. G , Smtth,
J A , and Struhl, K. (1995) Preparation of nuclear and cytoplasmic extracts from
mammalian cells, m Current Protocols zn Molecular Biology. Wrley, New York,
pp 12.1 I-12 1.9
16 Muhlemann, 0 , Kretvi, J.-P., and Akusjarvr, G (1995) Enhanced splrcmg of
nonconsensus 3 splice sites late during adenovtrus mfectron J Vzrol. 69,
7324-7327.
18
Adenovirus
217
and Protocols
NJ
218
IIIa and the hexon-cementmg protein IX, as well as the vertex protems penton
base and protruding fiber. Inside the nucleus of an infected cell, adenovnus
DNA is packaged mto the capsid together with condensing proteins V, VII, 1
p, and cysteine protease L3/p23.
1.2. Internalization and Penetration
Adenovirus type 2 uses a stepwise cell-entry program (8). Its fibers attach to
an epithelial-cell-surface receptor of the immunoglobulin gene family (9-11).
In the case of hematopoietic cells, an a, p2 integrm has also been reported as a
high affinity receptor attaching to the virus penton base protem (12). In epitheha1 cells, fibers are shed from the particle and vu-us enters by receptor-mediated endocytosis via tibronectm-bmding a& mtegrins binding to penton base
(13,14)). After 10 mm of mternalization, most vu-us particles are found
within endosomes. Penetration of mdividual virions across the endosomal
membrane is facilitated by slightly acidic pH and appears to require bmdmg of
penton base to the av& mtegrm (15,26). When added to cells m high amounts,
virions can also get into the cytoplasm m the absence of acidified endosomes
(14). In the cytoplasm, the internal capsid protein VI 1sdegraded by the reactivated viral protease and protein IX is dissociated from the capsid. Protease ts
reactivated by two signals, penton-base bmdmg to mtegrin receptors at the
plasma membrane and the reducing cytoplasmic milieu. These triggers prepare the capsid for disassembly near the nuclear membrane (17).
1.3. Nuclear Transport
All the proteins needed for virus assembly must enter the nucleus after synthesis m the cytoplasm. They either directly or indirectly contam nuclear targeting mformation, such as nuclear localization sequences (NLSs) Cfor revzew
of nuclear zmport ofprotezns, see ref. 18). It is possible that NLSs present on
capsid protein(s) are involved m nuclear targeting of incoming virus particles
In addition, mechamcal forces, generated by cytoplasmic motor proteins, may
be required for virus localization to the nucleus In any case, structurally
altered adenoviruses reach the nuclear membrane and attach to the cytoplasmic side of the nuclear-pore complex (14). Nuclear import of the DNA and
associated proteins, such as protein VII, then occurs after DNA is dissociated
from the capsid (17).
2. Materials
Commonly used chemicals were from Sigma (Buchs, Switzerland) or Fluka
(Buchs, Switzerland). All stock solutions were made up m double-distilled water
and filtered through 0.2~pm polyether sulfon membrane filters (Semadem,Switzerland). Working solutions were diluted mto double-distilled water before use.
Fluorescence Microscopy
219
2.1.2. Hardware
1 Cell mcubator, equilibrated to 5% humidified COZ atmosphere at 37C Rocking
plate fitting into Incubator.
2. Ultracentrifuge, SW 55 (or SW 50.1) rotor and correspondmg ultra clear thermoplastic tubes (Beckman)
3 Table-top centrifuge (Heraeus).
4 Low-speed centrtfuge holding 50- and 15-mL screw-capped plastic tubes.
5 Pasteur pipets, autoclaved.
220
Fluorescence Microscopy
221
2.3.2. Hardware
1 Round glass cover slips (I 2-mm diameter, Asststent, Winiger, Switzerland)*
Wash m 1 N hydrochloric acid for 10 mm (HCl), follow by three changes m
ddHzO, methanol, and agam ddH20.
2. Place between two filter papers m a glass Petri dish, autoclave, and dry in an
oven at 60C.
3 Alummntm plate.
4 Ice bucket
5. Wlppmg rocker kept at 4C
6 Reichert-Jung Polyvar microscope (Merck, Switzerland) equipped with a 40X
oil-Immersion obJectlve (numerical aperature 1.O), Nomarski differenttal interference optics (DIC) and a Texas red filter set (excttatton filter 530-585 nm,
emtsston filter LP 615) linked to a charge-coupled device (CCD) video camera
(Hamamatsu C5405, Hamamatsu Photomcs, Germany).
7. Macintosh computer (Quadra 650 Apple Computer, Cupertino, CA) online to the
camera system loaded with Argus-20 imaging acqutsitton program (Hamamatsu
Photomcs, Germany) and Photoshop Version 3 0.5 (Adobe).
8. NIH image-analysts software (version 1.6) (written by Wayne Rasband).
world wide web at http://1 28.23 1 98 16/nib-tmage/download.html.
3. Methods
3.1. A deno virus Purification
The following protocol was modified according to ref. 20.
1. Grow KB cells on ten 100~mm tissue-culture dishes in DMEM contaming 7%
Clone III serum to 90% confluency corresponding to approx 8 x lo7 cells.
2 Infect cells with l-5 plaque forming units (PFU) per cell in 3 mL of DMEMBSA on a rocking platform at 37C m a CO* incubator for 90 mm
3. Remove moculum, add 7 mL of DMEM-7% Clone III serum, and incubate m
CO2 Incubator at 37C until cytopathic effect (CPE) occurs approx 3 d later
Make sure CPE is complete and cells are detached from the dish.
222
4. Collect cells wrth a short Pasteur prpet and transfer to 50-mL screw-capped centrifuge tube. Pellet cells at 600g for 10 mm. Wash pellet tn 10 mL of PBS and
transfer suspensron to 15-mL centrifuge tube. Pellet cells
5. Resuspend cells In 0 01 M Trrs-HCl, pH 8 1, 0 5 mM PMSF at 2 x IO7
cells/ml and freeze-thaw three ttmes rn lrqurd nitrogen and a 37C water
bath At this point, the preparation can be kept frozen at -7OC for several
months.
6 Extract cells wtth an equal volume of Freon by gently vortexmg and shaking by
hand for l-2 min. The suspension should become viscous and homogenous Separate orgamc and aqueous phase by centrrfugatron at 1OOOgfor 5 mm at 4C and
collect upper-aqueous phase.
7. To prepare stock vuus, filter-sterilize aqueous phase through 0 45-pm drsk filter
and shock-freeze ahquots m liquid mtrogen. Store at -70C
8. Layer upper-aqueous phase on top of a CsCl step gradient and spin for 2 h at
32,000 r-pm m SW55 rotor (Beckman) at 4C (see Note 2)
9. Collect virus band by carefully removing liquid from the top using a Pasteur
prpet. Dilute virus (approx 400 &) with 0.01 M Trrs-HCl, pH 8.1, to 2 mL
and overlay over a second CsCl-step gradient as described m step 8. Spm
rsopycmcally for 18 h at 32,000 rpm at 4C.
IO. Collect vu-us band as descrrbed m step 9.
11 Vacuum-concentrate vuus m collodton membrane by dialysis agamst 0 01 M
Trrs-HCl, pH 8 1,0 15 MNaCl, 1 mMMgC12 Observe approx every 5 mm and
concentrate to l-2 mg/mL (approx 100-200 &). Disconnect vacuum and continue dialysis for another 2-3 h on ice. Change buffers at least twice Use vu-us
unmedlately or add glycerol to 10% and shock-freeze aliquots m hquid nitrogen
Store at -70C for up to several months
Fluorescence
Microscopy
223
3.2.4. SDS-PAGE
Vn-us homogeneity can also be tested by SDS-polyacrylamide gel electrophoresls (SDS-PAGE) followed by Coomassle blue staining, e.g., as described
224
of Adenovirus
3.3.2. Cold-Synchronized
1 Two days before infection, seed HeLa cells (or any other cell line) on round glass
cover slips in DMEM growth medium placed m a 24-well dish At the day of
infection, cells should be 60-90% confluent
2. Transfer 24-well dish to an aluminum plate coated with a wet ktmwipe and kept
on ice. Wash cells once with cold-binding medium (RPMI-0 2% BSA, 15 rtuV
Hepes-NaOH, pH 7 4).
3. Dilute stock Texas red labeled virus (typically 0 2 $ of 0 26 mg/mL per cover
slip, approx 1,000 vnus particles per cell) into cold-binding medmm and add 0 2 mL
of this dilution to each cover slip.
4. Bind virus to the cell surface m the cold on a gently rocking platform for 90 mm
5. Wash off unbound virus with cold-binding medium and briefly add 0.5 mL of
warm internalization medium (DMEM-0.2% BSA) to each cover slip Remove
medium and add fresh 0 5 mL of warm DMEM-0.2% BSA Incubate for a given
time in a 37C CO, incubator
6 Wash cells quickly in PBS at room temperature and fix immediately m 3.3%
paraformaldehyde for 15 min at room temperature
7. Quench unreacted paraformaldehyde with 50 ti NH4Cl m PBS for 5 mm at
room temperature and wash briefly with PBS (see Note 7)
8. Mount slide in PBS contammg DABCO and seal edges of cover slip with nail
polish (see Notes 8 and 9)
9. For best results, analyze within 24 h
225
Fluorescence Microscopy
FITC
MW
- 31
- 22
- 14
-
Fluorogram
Total Protein
226
Nom
Ad-txred
37C
60
Fig. 2. Entry of Texas-red-labeled adenovirus into HeLa cells. Virus was bound to
the cell surface in the cold for 90 min (A,B) and internalized for 10 min (C,D) or 60 min at
37C (E,F). Nomarski DIC optics was used to visualize the cells (Nom). Virus was
detected by fluorescence microscopy using the Texas red filter set (Ad-tx red). The focus
of the microscope objective was set to the middle of the cells. The apparent depth of
field was estimated to be in the order of several pm (see ref. 26). Outlines of cells and
nuclei in panels B, D, and F were generated with the NIH image software program.
10. Under Special, define image to register and the DIC outline image to register.
11. Use the Blend and Divide commands to merge the images.
12. Define the Density Slice such that all the fluorescent particles become colored
and are selected. Save image.
13. Outline the area of interest (nucleus or whole cell) using the free hand mode
and determine the number of fluorescent particles by the Analyze Particles function.
14. Import data into a statistics program (e.g., Microsofts Excel or Adelbecks
KaleidaGraph) for further analysis and data presentation (for example, see Fig. 3).
15. Process TIFF files and arrange with Adobe Photoshop software program for printing, e.g., on a Fuji Pictography 3000 sublimation printer.
As can be seen in Fig. 2, virus bound to the cell surface of HeLa cells in the
cold is more or less evenly distributed over the cell. The ratio of virus particles
over the nucleus compared to the cytoplasm (NucKyt) is approx 0.29 (Fig. 3).
After 10 min of virus internalization at 37C, a qualitatively similar result is
227
Fluorescence Microscopy
To.32
b
TI) 0.2
Z
0
OC, 90 min
37%,
10 min
37C, 60 min
of viruses has now moved toward the nucleus. The nuclear-to-cytoplasmic distribution coefficient at 60 min of warming cells is about 1.28 (Fig. 3).
It is important to note that by conventional fluorescence microscopy it is not
possible to determine how close to the nuclear membrane the virus particles in
fact are. Confocal microscopy and thin-section electron microscopy have been
applied to resolve this question. The results will be presented elsewhere demonstrating that the fluorescently labeled viruses enter the cells and localize to
nuclear pore complexes with similar efficiencies as unlabeled control virus
(Greber et al., submitted). No aggregated viruses inside or outside the cells are
detected by electron microscopy, suggesting that each fluorescent dot represents a single virus particle. Thus, the method described here truly measures
virus transport from the cell surface or endosomes across the cytoplasm toward
the nucleus.
4. Notes
1. Typical yields of virus vary from cell to cell and of course depend on the number
of cells on the dish. We found that ten 100~mm dishes of KB cells (approx 4 x lo7
cells) yield approx 1 mg virus.
2. Freon helps dissociate aggregated virions during virus extraction from the cells.
To prevent virus aggregation after cell lysis, it is important not to overload the
228
3
4.
7
8.
9.
CsCl gradients. One SW50 tube should not contam more than 0.5 mg of vtrus
For long-term storage, a concentration of 2 mg/mL vtrus should not be exceeded
Check pH of the silicotungstic actd stock solutton each time before use (pH will
drop with time).
If the adenovirus preparation 1scontaminated with AAV, at leastone strong band
of AAV protein 3 (VP-3) shows up below the adenovnus I&/fiber bands in SDSPAGE (data not shown; see also ref. 25). AAV proteins 1 and 2 (VP- 1 and VP-2)
are less abundant than VP-3 and stain weakly m Coomassie blue.
Texas-red labeled virus prepared under these conditions contams maximally 2 4
molecules of Texas red per hexon monomer. Texas-red labeled vuus has the same
specific mfectivity (per protem) as unlabeled virus as determined by plaque assay This vnus preparation remains active for months
Fluorescem tsothiocyanate (FITC)-labeled adenovtrus can be prepared using
stmtlar protocol Dialyze virus against 0 01 h4 sodium bicarbonate contaimng
0 14 MNaCl, 1 mMMgCI,, for 2 h and determme the protein concentratton using
Micro BCA assay (Pierce) Adjust the pH of the vtrus solution to 9 0 by adding l/5 of the volume 0 5 Msodmm carbonate buffer, pH 9 0 Initiate labeling by
adding 16 p.L of 0 5 mg/mL FITC solution (dduted tmmedtately before use mto
0 1 M sodium carbonate buffer, pH 9 0 from a 5-mg/mL stock solution made m
DMSO) to 125 pL of 1.8 mg/mL virus suspension Incubate for 1 h m the dark
on a rocker at room temperature Treat with hydroxylamrne and repurtfy virus on
CsCl gradient as descrtbed above Analysis of FITC-labeled vu-us by SDS-PAGE
and fluorotmagmg Indicated that 47% of the label 1s mcorporated into hexon,
13% into penton base, 18% into protem IIIa and fiber, 3% into protein VI, 7%
mto protein VII, and 12% mto protein IX (Fig. 2, lane 2)
PBS contammg 0.1% sodium azide can be used Instead of PBS to mmlmtze bacterial contammation.
DABCO-contammg mounting media only lasts a few weeks and should be made
fresh on a regular basts An alternative mounting medmm is based on NPGT
(N-Propyl-gallate-Glycerol-T&)
For 25 mL, mtx 17 5 mL glycerol (87%), 7.5 mL
0.1 M Tris-HCI, pH 9 5, 2.5 g N-propyl-gallate m a 50-mL screw-cap plastic
tube Place tube into water bath sonicator (e g., Branson Model 12 10) and somcate
for 15 mm at 37C. Transfer solution into vacuum flask and degas extensively
under house vacuum. Store ahquots at -20C. NPGT medium IS recommended
for Texas-red- or rhodamme-labeled probes, but appears to quench fluorescemlabeled probes
A third mounting medium consists of 80% glycerol, 20 mA4 Trts-HCl, pH 8 8,
0.5% Paraphenylene-diamine (Sigma). This medmm has a slightly brownish color
and lasts several months tf stored at -20C. It works equally well with both Texas
red and FITC probes.
Acknowledgments
We thank Dr. Robert Stldwlll for help with image acqulsltion and analysis
and comments to the manuscript. This work was supported by the SWISS
Fluorescence
Microscopy
229
National Science Foundation (grant no. 3 1-43412.95 to UFG) and the Kanton
of Zurich.
References
1 Mulligan, R. C (1993) The basic science of gene therapy. Scrence 260,926932.
2. Trapnell, B. C. and Gorziglia, M. (1995) Gene therapy using adenoviral vectors
Curr. Op Btotech 5,617-625.
3. Hanama, E G., Kavanagh, J , Hortobagyi, G., Giles, R E., Champlm, R., and
Deisseroth, A B. (1995) Recent advances in the applicatton of gene therapy to
human disease Am J Med 99,537-552.
4 Rosenfeld, M A. and Collins, F S. (1996) Gene therapy for cystic fibrosts Chest
109,241-252
5. Crystal, R G. (1995) Transfer of genes to humans: early lessons and obstacles to
230
17.
18.
19.
20.
21.
22.
23
24.
25
26
19
Simultaneous
Detection of RNA and Proteins
in Adenovirus-Infected
Cells by Fluorescence
/II Situ Hybridization (FISH) and lmmunostaining
Eileen Bridge
1. Introduction
Adenovnus (Ad)-infected cells have been used extensively as a model system for studying the expression of eukaryotic genes and were instrumental m
elucidatmg steps m the production of RNA. Much of this work involved genetic
and molecular analyses of viral gene expression. Recent advances m methodologies for detecting nucleic acid and protein components of cells has triggered a renaissance m the study of cellular organization. Cell biologists
interested m understandmg functional organization of gene-expression activtties are again turnmg to Ad-infected cells as an important model system
(reviewed m ref. 1).
A major goal of current research in cell biology is to understand how repltcation and gene-expression activtttes are organized relative to each other and
to the structural framework of the nucleus, The location of transcription and
replication activities in Ad-infected cells has been studied by pulse-labeling
cells with trttiated nucleotides followed by autoradiography and electron
microscopy (2,3). Methods for mcorporation of modified nucleotides followed
by fluorescence-based detection have also been developed (4,5), and allow for
simultaneous detection of rephcation and transcription activities. Localtzatton
of posttranscriptional events such as splicing relies on mnnunostaining techniques to determine the localization of the factors involved m catalyzing these
reactions, and on in situ hybridizations (ISHs) to determine the localization of
precursor and product forms of the RNA (6,7). RNA export involves movement of RNA from the location of its transcription to the nuclear pore, where It
From
Methods
m Molecular
Medmne,
Vol
21
231
Adenovrrus
Methods
and Protocols
NJ
232
Bridge
is transferred to the cytoplasmic compartment, and 1slikely to involve interactlon of the RNA wtth protein carriers (8,9). A combination of ISH and
nnmunostammg to simultaneously determme the locallzatlon of both specific
nucleic acids and specific proteins can thus be a powerful method for studying
their relationship within the organizational framework of the cell.
In vu-us-infected cells, localrzatlon studies have been used to determine the
distribution of viral and cellular proteins relative to sites of viral-DNA accumulation (10-13). In particular, Bosher et al. (II) were able to demonstrate
that nuclear factor l(NF1) IS present m viral replication factories produced m
cells after infection wtth Ad2 but not after mfection with Ad4. Since NFI is an
essential replication factor for Ad2 strains but not needed for rephcatlon of
Ad4 strams, this study demonstrated nicely that the locahzatlon of this cellular
factor in viral rephcatlon factories was likely to be the result of its function in
viral rephcatlon. Localization of RNA relative to cellular and vu-al proteins has
also been studied by ISH techniques (S-7,14-19). We have used a combmation of immunostainmg and ISH to study the localization of viral RNA relative
to nuclear structures that contam sphcmg factors (14). Localization studies such
as those described above require careful attention to a number of different
parameters. These include fixation and pretreatment of cells, preparation of
suitable probes, and hybridization conditions.
Each of these parameters will be discussed separately in the sections below.
1.1. Fixaiion and Pretreatment of Cells
We have routinely grown and Infected cells on glass shdes or cover slips. At
appropriate times after infection, the cells must be treated with a fixative to
preserve structure, and permeabilized to allow accessof probes and antibodies
to their target molecules. Common fixatives include crosslmkmg agents such
as paraformaldehyde, formaldehyde, or glutaraldehyde. Fixed cells can be
permeabllized by extraction with buffers contammg detergents such as Triton
X-100, nonidet P-40 (NP40), saponin, and dlgltonin (20) or mild treatments
with sodium dodecyl sulfate (SDS) (5) to allow access of probes and antlbodies to target molecules. Alternatively, fixing with agents such as methanol or acetone can simultaneously fix cellular structure and permeabihze
the cells to allow access to probes. The choice of treatments for fixmg and
extracting cells varies greatly and can critically affect the outcome of the
experiment. Unfortunately there 1sno single protocol that works reliably for
many antibodies, cell types, and probes. We have found it necessary to titrate
both fixation and extraction conditions in order to obtain the best staining for
each combination of antibody and in situ probes. In every case it is necessary
to balance the need for good accesslbllity of target molecules with the best
preservation of nuclear structure.
233
234
Bridge
serve as a useful marker for the location of single-stranded DNA during the
late phase of Ad infection. In this article I will focus on the methodology we
have used for locahzing viral RNA and cellular or viral proteins in doublelabeling experiments.
2. Materials
Note All solutions and reagents must be kept RNase free for the detection
of RNA.
I Btotm- 16-dUTP (Boehringer Mannheim, Mannhelm, Germany)
2 G-50 Sephadex spm chromatography G-50 Sephadex from Pharmacta (Uppsala,
Sweden) IS prepared for use accordmg to the manufacturers mstructions and usmg
precautions to prevent contammation with RNases We routmely autoclave the
hydrated G-50 sephadex and store it at 4C.
3 Glass cover slips. We use 12 x 12-mm glass cover slips and prepare them by
washing in 70% ethanol, and then dtstrtbutmg the cover shps between layers of
Whatman 3MM paper in a glass Petrt dish The cover slips m the Petri dish are
autoclaved and are then ready to use.
20X SSC Rectpe for 1 L of 20X SSC 175 3 g NaCl, 88 2 g sodium citrate.
Adjust to pH 7.4 with NaOH. Autoclave.
Phosphate buffered salme (PBS) Recipe for 1 L 8 0 g NaCl, 0 2 g KCl, 1 44 g,
Na2HP04, 0.24 g KH2P04. Adjust to pH 7.4 with HCl Autoclave.
4 0% paraformaldehyde (PFA) m PBS. MIX PFA with PBS While stirring add
NaOH until the paraformaldehyde goes into solutron Filter through a 0.2-w
filter. PFA can be frozen m aliquots at-20C for long-term storage or kept at 4C
and used within 2-3 wk
7. 0 5% Trrton X-100 m PBS. This solutron 1s filtered through a 0 2-w filter and
stored at 4C
8 Hybndizanon soluuon: 50% detomzed formamtde, 2X SSC, 5% dextran sulfate, 50 mM
phosphate buffer, pH 7.0,l mg/mLE ~011t-RNA (Stgma, St Louis, MO). Store at-20C
9 Hybridizatton chamber. A moist chamber 1s required to prevent the cells from
drying out during hybridization Our homemade chambers consist of a box that
contains paper towels wetted with 50% formamlde, 2X SSC. Slides contammg
the cover slips are placed on stands within the box, and the lid sealed with
parafilm before placing at the appropriate temperature
10. Blockmg solution* 0 5% Blocking reagent from Boehrmger Mannheim m 100 nnI4
Trrs-HCl, pH 7.5, 150 mMNaC1 Autoclave the solution to dissolve the blocking
reagent. Alrquot and freeze at -20C
11 Tween-20
12. ExtrAvidm-FITC
(fluorescein tsothlocyanate) (Sigma).
13 Primary anttbody against the protein of interest
14. Secondary antibody conjugated to Texas red or other appropriate fluorochrome
15. Mounting Medium Vecta-Shield from Vector Labs, (Burlingame, CA) This contains antifadmg reagents to preserve the fluorescence during microscopy.
235
of Probes
3.2. Preparation
and fixation
of Cells
5 Wash cells twice for 5 mm at room temperature with 2.0 mL 2X SSC; they are
then ready for ISH
1. For each 12 x 12-mm cover slip, dry down approx 50 ng of labeled PCR product
m a speed vat (the optimal amount of probe can be determined by titration).
Dissolve the probe m 8 $ of hybrldlzatlon solution.
2 Denature the probe by heating to 70C for 5 mm. Chill on ice.
3. Plpet 8 & of denatured probe onto a clean glass slide. Lift out a cover slip with
forceps, blot the back of the cover slip dry with a tissue, and then carefully layer
the cover slip cell-side down onto the drop of hybrldlzation solution contaming
Bridge
236
4.
5.
7
8.
9.
10
11.
12
the probe Incubate the slide and the cover sltp m a hybridization chamber equthbrated with 50% formamide, 2X SSC at 37C for 1 to 3 h.
Loosen the cover slip by ptpetmg 2X SSC around the edges Gently pry up the
cover slip usmg a needle and forceps and transfer tt cell-srde up to a 3.5-cm dish
containing 2X SSC Wash m 2X SSC for 3X 15 mm at 37C. Wash m 1X SSC for
15 mm at room temperature (see Note 3)
Rinse cover slips with 4X SSC contammg 0.05% Tween-20 Aspirate thts
buffer, taking care to dry around the edges of the cover sltp and then add 8 p.L
of blocking solution to the top of each cover slip Incubate for 30 min at
room temperature.
Rmse cover slips with 4X SSC containing 0.05% Tween-20. Aspirate, add 8 u.L
of FITC-coupled extravidm (Sigma) diluted l/200 m blockmg solutton ( 12 5 &mL
final concentratron), to the top of each cover slip, and incubate for 30 mm at
room temperature. When performing double stammgs in which ISHs are performed together with rmmunostaming, it is convenient to incubate the primary
antibody together with the extravtdm at an approprrate dtlutton (see Note 4).
Wash cover slips three times for 5 mm with 4X SSC at room temperature
Rmse cover slips with 4X SSC contammg 0 05% Tween-20 Aspirate, add 8 pL
of an appropriate secondary antrbody coupled to Texas red (Southern
Biochemicals, Birmmgham, AL) diluted l/50 in blocking solutron (final concentration 0.02 mg/mL) Incubate the cover slip at room temperature for 30 mm
Wash cover slips three times for 5 mm with 4X SSC at room temperature.
Postfix the cells with 4% paraformaldehyde m PBS for 5 mm at room temperature
Wash the cells three times for 5 mm with PBS at room temperature (see Note 5)
Mount the cover slips onto a slide contammg a 3.5)Ic spot of Vecta-shield
mounting medmm, seal with nail polish, and then observe the stammg by fluorescence mtcroscopy using appropriate filters for the two different fluorochromes
237
the cover shp Incubate the dish in a moist chamber (a covered box containing
paper towels soaked m water) at 37C for 45 mm.
3 Wash the cover slips three ttmes for 10 min with 2X SSC at room temperature
4. Proceed with the hybrtdtzation protocol (Subheading 3.3.1.).
4. Notes
1. The procedure for fixing and permeabilizing the cells to both preserve structure
and allow access to macromolecular reagents is highly variable We have found
it necessary to titrate the amount of paraformaldehyde used for fixation and usually get good results with 14% paraformaldehyde solutions. We try to use as
close to 4% paraformaldehyde as possible in order to maintain the best preservation of structure Pre-extraction of the cells with 0 5% Trtton X-100 m PBS for
30 s to 3 min on ice prior to fixation with paraformaldehyde can help to increase
permeablhty of the fixed cells, but can also result in loss of cytoplasmic RNA
(22). We have used a mild treatment with sodium dodecyl sulfate (SDS)-containmg buffers ($14) to allow greater access of probes to the cytoplasmic RNA In
this procedure, incubate cells for 5 min at room temperature in buffer containing
0.1% SDS, 100 mA4Tris-HCl, pH 7.5, 150mMNaC1, 12 5 mMEDTA, followmg
238
Bridge
fixation m 4% paraformaldehyde. This step replaces the extractton with 0 5%
Trlton X-100 containing buffer. It is advisable to titrate the amount of SDS m the
solution. We have used a range between 0.05 and 0 2% Detection of cytoplasmic
RNA may require additional treatments with protease to remove the proteins and
allow access of the probes to the RNA (28). We have tried to avold this step since
we are specifically interested m looking at the localization of both protems and
RNA, but there may be circumstances where protease treatments are desirable.
Storage of cover slips followmg fixation. We have observed the best staining of
RNA ISHs usmg cells that are fixed and permeabllized and then used directly for
the ISH protocol. However several laboratories obtain good results when fixed
cells on cover slips are stored m 70% ethanol and then used for ISH within several months (6,7,15,22).
Hybridization and washing conditions may require titration We have done the
hybrldlzatlon at temperatures ranging from 37 to 55C, and washing temperatures have ranged from room temperature to 42C. Where viral RNA IS being
detected the goal 1s to optimize the signal-to-noise ratlo between infected and
unmfected cells
ISH is a relatively harsh procedure; we have encountered several instances m
which an antibody no longer detects Its antigen following the ISH protocol. This
1s presumably because the ISH procedure affects the epltope recognized by the
antlbody. It can be useful to test several lmmunologlcal reagents against the protem of interest to find one that still efficiently recognizes the protein followmg
ISH It may also be helpful to perform the antlbody immunodetectlon first followed by the ISH protocol (28).
Subheading 3.3., steps 10 and 11 are optional, but I have found that the ISH
signal remains stable for a longer period of time if the cells are postfixed followmg the ISH protocol
References
1. Bridge, E and Pettersson, U. (1996) Nuclear organization of adenovlrus RNA
btogenesls Exp. Cell Res 229,233-239
2 Puvlon-Dutilleul,
F. and Puv~on, E (1991) Sites of transcription of adenovirus
type 5 genomes m relation to early viral DNA replication m infected HeLa cells
A high resolution ISH and autoradlographlcal study &ol. Cell 71, 135-147
3 Puvlon-Dutilleul,
F. and Puvlon, E (1990) Rephcatmg single-stranded adenovlrus type 5 DNA molecules accumulate within well-delimited mtranuclear areas of
lytically infected HeLa cells. Eur J Cell Bzol 52, 379-388
4. Jackson, D. A , Hassan, A B., Errington, R. J , and Cook, P R. (1993) Vlsualizatlon of focal sites of transcription within human nuclei EMBOJ 12, 1059-1065
5. Pombo, A., Ferrelra, J., Bridge, E., and Carmo-Fonseca, M. (1994) Adenovnus
replication and transcription sites are spatially separated in the nucleus of Infected
cells. EMBO J. 13,5075-5085.
6. Xmg, Y., Johnson, C V., Dobner, P. R., and Lawrence, J. B. (1993) Higher level organization of indlvldual gene transcription and RNA splicing. Science 259, 1326-l 329
239
23. Challberg, M. and Kelly, T. (1989) Animal virus DNA replication. Ann Rev
Biochem 58,67 l-7 17
24. Puvion-Dutilleul,
F and Prchard, E (1992) Segregation of viral double-stranded
and single-stranded DNA molecules m nuclei of adenovnus-infected cells as
revealed by electron microscope ISH Blol Cell 76, 139-150.
240
Bridge
25 Puvlon-Dutilleul,
F and Puvion, E. (1990) Analysis by ISH and autoradiography
of sites of replication and storage of single- and double-stranded adenovnus type
5 DNA m lytically infected HeLa cells J Struct Biol 103,280-289.
26. Puvron-Dutllleul,
F., Pedron, J., and Cajean-Feroldi, C (1984) Identlficatlon of
mtranuclear structures containing the 72K DNA-bmdmg protein of human adenovms type 5. Eur J. Cell Blol. 34, 3 13-322
27 Sambrook, J., Fritsch, E. F., and Maniatis, T (1989) Molecular Clonzng. A Laboratory Manual, 2nd ed Cold Spring Harbor Laboratory, Cold Sprmg Harbor, NY.
28. Dnks, R. W., van de Ryke, F. M., Fujtshtta, S., van der Ploeg, M., and Raap, A. K
(1993) Methodologies for specific intron and exon RNA locallzatton m cultured
cells by haptemzed and fluorochromrzed probes. J Cell Scz 104, I 187-l 197
20
Characterization
Jeffrey
of the Adenovirus
Fiber Protein
1. Introduction
Entry of the vu-us particle during the early stages of infectton IS a multtstep
process that includes: initial attachment of the virus capsid via a highaffinity interaction with a cell-surface receptor protein (Z-4); a second lowaffmrty interaction of the penton-base protein in the capstd with avP3 and/
or avP5 integrms (5-7); and internahzation of the vnus capstd mto early
endosomes (3).
With the recent interest m the use of adenovnus for transfer of genetic mformatron for therapeuttc benefit (adenovirus gene therapy) (a), studies of the
structure and function of the fiber protein have enjoyed a renewed populartty,
smce this protein mediates the initial attachment of the virus capstd to a cellsurface receptor (Z-4). The fiber protein IS a complex of three apparently identical subumts that IS found at each of the vertices m the icosahedral virus
particle (9); by sodium dodecyl sulfate-polyacrylamtde gel electrophorests
(SDS-PAGE), the molecular weights of the monomertc and the trimeric form
of human adenovnus type 2 (Ad2) and Ad5 fiber are 62,000 and approx
lSO,OOO-210,000, respectrvely (9,IO). This trtmeric structure has been proposed to consist of (refs. 2 and II; Fig. 1): an N-terminal domain that interacts
with the vnus penton base and contains signals for transport of the protein to
the nucleus, a shaft that has been suggested to contain 15 residue repeating
mottfs, and a C-terminal knob that mediates the attachment of the protein complex to the cell-surface receptor. This C-terminal knob also contains determtnants for type-specific antigens.
An alternative left-handed triple helrcal model for the shaft has also been
proposed (12). The recent report of the structure of a C-terminal knob
From
241
and Protocols
NJ
242
PENTON
BASE
domain for Ad5 fiber is consistent with this structural organization for the
fiber trlmer (13,1#).
In other serotypes of fiber, the apparent lengths of the trlmers are different,
based on electron-mlcroscopy measurements and comparison of the sequences
of the fiber genes from different serotypes (1548); this difference is generally
attributed to the number of repeatmg motifs found in the shaft domain In several serotypes of adenovn-us, the virion encodes two fiber proteins, but the
reason for the presence of these additional fibers IS not yet known. The adenov~rus fiber protein from serotypes 2, 5, and 12 1s also known to contam a
posttranslational modification, 0-lmked N-acetyl-glucosamme (O-GlcNAc;
J9-22), which IS believed to be added by the action of a cytoplasmtc enzyme
prior to entry of the protein m the nucleus.
In order to study the structure and function of fiber and of fiber mutants, a
number of strategies have been employed.
1, A number of laboratorles have studied temperature-sensitive mutants m the Ad2
and Ad5 fiber gene (for example, refs. 20 and 23). Temperature-sensltlve mutations (for example, H.5~142) that affected trlmer formation and that affected the
ability to be labeled by 3H-glucosamme (and presumably deficient m the addltlon
of 0-GlcNAc) were identified by this approach.
2 Expression of mutant-fiber protein has also been achieved m heterologous
expression systems, such as E co11 (24), baculovlrus (25), and vaccmla virus
systems (26,27) Using site-directed mutagenesis, specific mutations m the
fiber gene can be constructed and the resultmg gene product studied for alteration of Its structure and/or function An advantage of these systems IS that
production of the fiber protem does not depend on the necessity of formlng
an Infectious adenovlrus capsld for propagation of the mutant protem,
although adenovmons lackmg fiber can be produced and appear to be somewhat
mfectlous (28).
Adenovirus
fiber Protern
243
2. Materials
and Hydroxylapatite
1. Vu-uses and cells. Ad2 and Ad5 vtruses can be grown on plates of human HeLa
(ATCC #CCL2) or other permissive cell lines (A549, ATCC, Rockville, MD,
CCL185; KB, ATCC CCL17, and 293, ATCC CRL1573) Spmner culture of
human HeLa cells (HeLa S3, ATCC CCL2.21) can also be used. Grow HeLa cells
m S-MEM (Gibco-BRL, Gaithersburg, MD) with 7% bovine calf serum. Generally mfecttons for preparatton of fiber protein are performed at multtplicity of
infectton (MOI) of approx 2&30 PFU/cell. There are several protocols available
for preparation of vuus from infection, such as the one used m Subheading 3.1.1.
2 Phosphate buffered Saline (PBS), pH 7.3-7.4. 137 r&I NaCl, 2 7 mM KCl, 4.3
m&I Na2HP0,*7H,0,
1 4 mM KH2P04
3. 10 mMTrts-HCl,
pH 8 1, 1 ti EDTA.
4. Freon 113.
5. CsCl, density 1 4 g/mL (see Note 2), and CsCl, density 1.2 g/mL
6 Boiled dtalysis tubmg.
7 10 mM Sodium phosphate buffer, pH 7.4 at 4C
8 Saturated ammonmm sulfate solutton at 4C.
9 DEAE-Sepharose column equilibrated with 10 and 50 m&I sodmm phosphate
buffer, pH 6.8 at 4C
10. 0.5 MNaCl
1 1 Hydroxyapattte column equdibrated wtth 10 tipotassium
phosphate buffer, pH 6 8
12 Apparatus and reagents for SDS-PAGE
13 Apparatus and reagents for Western blot
of Infected Cells
244
8 Ice-cold methanol
9 Antibodies available. Several polyclonal and monoclonal antibodies (PAbs and
MAbs) are available for studtes on adenovn-us fiber. PAbs such as R72 (agamst
Ad2 full-length fiber; ref. 29) or RAF2Nat (against nondenatured Ad2 full-length
fiber; ref. 25) are useful for tmmunoprectpttations or Western blots of mutant
fiber polypepttdes, antibody RaF2Nat is also useful for immunofluorescence of
Ad2 fiber truncatton mutants. PAbs against virton components (available from
the Amerrcan Type Culture Collectton) may also be useful in some cases, but
generally will recogmze all three maJor capsrd protems (hexon, penton base, and
fiber). Alternatively, a large number of MAbs are available. Several MAbs made
m our laboratory have proven very useful for structural studtes of fiber by Western blot and by mdnect tmmunofluorescence.
a 4D2-5 recogmzes both monomer and trimer fiber protems of Ad2, Ad5, and
Ad7 (26); the epttope lies between residues 10 and 17 (30)
b 2A6-36 recognizes only the trimeric form of the polypeptide and recogmzes
an epitope in the shaft of the trimer; the epttope for this antibody is located
between residues 60 and 260.
Other antibodtes that are type specific and that recognize other domains m
the protem have also been described; for example, J Chroboczek and colleagues recently reported a series of monoclonal antibodies made against Ad2
fiber knob (32)
10. Mouse nommmune IgG (control)
11. FlTC-labeled goat anttmouse IgG or other fluorescent secondary antibody.
12. Hoechst 33258: 20 pg/mL m PBS (optional)
13 Cover slip mounting medium (Vectashteld, Vector Lab, Burlmgame, CA)* 0 1%
p-phenylene diamme m 9.1 glycerol/PBS (see Note 8 for recipe)
14 Nat1 polish.
2.3. Detection
of Fiber Monomers
2.4. Detection
of 0-GlcNAc
Addition
to Fiber
Adenovirus
245
Fiber Protein
3. Methods
3.1. Purification
Cells
and Hydroxyapatite
Infection of human HeLa or other permissive human cell types by Ad2 and
Ad5 leads to production of a large fraction of capsid proteins (including fiber)
that are not mcorporated mto vtrions. This protocol 1smodified from that of
Boulanger and Puvton (32) and is a modtfication of earlier protocols from
Phtllipson (33) and Maize1 et al. (34). The reference for purification of adenovirus parttcles and unmcorporated
(see also Note 1).
(virions). The supernatantabove the vu-ion band is the source of adenovirussoluble fiber. Remove this band from the tube with a syringe and needle or with
careful pipettmg. (For Isolating banded virus, two C&l separations are recommended The supernatant above the virus band from each separation may be
pooled and used m the next steps.)
9. Transfer the band to a piece of boiled dtalysis tubmg and dialyze the sample
agamst 3 to 4 changes of 10 mM sodium phosphate buffer (pH 7.4) at 4C to
246
Lipids
Empty Capslds
Adenovirus
components
viral coat protems
particles
10.
11
12
13
14.
15.
16
17
18.
19
remove the CsCl from the solutron Each change of buffer should contam l-4 L
of solution.
Slowly add a saturated solutton of ammonium sulfate at 4C to a final concentration of 55% ammonium sulfate. Allow the ammonmm sulfate prectpttate to form
for 15 h at 4C and pH 6.5.
Spm the precipitate at 500013 for 20 mm at 4C.
Dtssolve the pellet m a minimal amount of 50 mM sodmm phosphate buffer,
pH 6.8, at 4C
Dialyze the sample at 4C agamst 10 volumes of 50 mA4 sodmm phosphate buffer
(pH 6.8) over 72 h wrth at least five changes of buffer.
After dialysis, centrifuge the solution at 110,OOOg for 2 h This supernatant 1s
ready for column purtficatlon or for storage at -20C (see Note 3)
Load the sample onto a DEAE-Sepharose column equilibrated with 50 mM
sodmm phosphate buffer, pH 6 8, at 4C
Wash the column with at least 10 column volumes of 50 mM sodium phosphate
buffer, pH 6 8, at 4C.
Elute the column with a linear gradient of sodium chlortde (from 0.0 to 0 5 M), m
50 mM sodium phosphate buffer (pH 6.8). Collect fractions and assay for the
fractions contaming protein by absorbance at 278 nm
Fractions containing fiber are tdentlfied by SDS-PAGE of altquots of fracttons on a 10% gel Fractions containing fiber can be tdentrfred by Western
blot of the gel, usmg an antrbody specific for fiber (protocol given m Subheading 3.3.)
Pool the fractrons containing fiber protein and dialyze the pooled fractions against
10 mM potassium phosphate, pH 6.8 (see Note 4)
Adenovirus
247
Fiber Protein
20. Load this dialyzed solution onto a hydroxyapatite column equilibrated with 10 mJ4
potassium phosphate buffer, pH 6.8.
21. Wash the column with at least 10 column volumes of buffer.
22. Elute the fiber protein with a linear gradient of 0.01-O 50 M potassium phosphate, pH 6 8
23. Collect fractions and test for protein concentration and the presence of fiber as
described m step 18
of Infected Cells
Indirect lmmunofluorescence
of infected cells provides one of the most
direct means for assessing the expresston of fiber and fiber mutants wlthin
Infected cells. With the use of appropriate conformation-specific
antibodles,
248
information
about the ability
cell can also be determmed
1. Grow the cells to be infected on cover slips that can be viewed m an appropriate
hght microscope. This growth is most easily accomplished by placmg sterile
cover slips in the bottom of a 35-mm trssue-culture dash, mto which the cells will
be seeded in appropriate medium (DMEM + 5% fetal bovine serum, see Note 5)
The cells should be seeded at least 24-36 h prior to mfection, to msure that the
infected cells remain attached to the cover shp see Note 6.
2 When 60-80% confluent, infect the cells with adenovu-us (MO1 approx 10) or
other recombmant vu-us that expresses fiber
3. After 24-60 h of mfectlon, wash the cells on the cover slip once with phosphatebuffered salme (PBS) and fix them with 3% paraformaldehyde in PBS for 10-l 5 mm
at room temperature
4 Wash the cells with Trrs-buffered salme (TBS) or with PBS Do not allow the
cells to dry during the procedure
5. Incubate the cells on the cover slip with TBS or PBS contammg 1% Triton X- 100
(TBS-TX or PBS-TX-BSA) to permeabihze the cells for 10 mm at room temperature. As an alternative, the cells can be also permeabihzed by treating them
with ice-cold methanol for 2 min
6 Block nonspectfic bmdmg with TBS-TX or PBS-TX supplemented with 1% BSA
(TBS-TX-BSA or PBS-TX-BSA) in the same buffer for 30 mm at room temperature
7 Incubate the cells wrth a I:100 to 1:500 dtlutron of antrbody (ascites fluid of
either 4D2-5 or 2A6-36, the appropriate dilution should be determined experimentally) for 1 h at room temperature or 37C m a humidified chamber Control
IgG should be used as a negative control. If you use tissue-culture supernatant
taken from hybridoma cells, use it without further dilution (see Note 7)
8 Wash the cells with TBS-TX-BSA or PBS-TX-BSA for 15-30 mm with three
changes of buffer.
9 Add an appropriate dilution (approx 1: 100, but the approprrate dilution should be
determmed experimentally) of FITC-labeled goat antimouse Ig as a secondary
antibody for 1 h at room temperature.
10 The cells on the cover slip are then washed with three changes of TBS-TX-BSA
or PBS-TX-BSA (5 min per wash).
11. Optional* If you need to specifically visualize the nucleus, treat the cells with
Hoechst 33258 (20 pg/mL rn PBS) for 4 mm at room temperature to stam the
nucleus. Rinse the cells on the cover slips briefly with TBS or PBS.
12. Mount the cover slips on slides using mounting medium (see Note 8) Remove
any excess mounting medium by absorbing with a small prece of filter paper (the
bigger the pore size, the better)
13. Seal the edges of the cover shps wrth nail pohsh Allow the polish to dry thoroughly before proceedmg further Protect the cover slips from light by covering
with aluminum foil while drying.
14 View the infected cells usmg a Nlkon Opttphot mtcroscope equipped for fluorescent illumination or similar equipment
249
I5 The slidescan be stored at -20C but they must be kept m an opaque shde box or
wrapped m foil to minimize
exposure to light.
3.3. Defection
ern blotting
Fiber
250
A
123456
123456
-+
T2,5-@
M7+
Fig. 3. Western blot of fiber monomers and trimers. Aliquots of cell lysates from
Ad2-, Ad5-, and Ad7-infected A549 cells were separated by SDS-PAGE on a 10% gel
and blotted onto Immobilon membrane, prior to developing with monoclonal antibody
4D2-5 (A) or 2A6-36 (B). The sizes expected for monomers (M) and trimers (T) of the
various molecules are indicated by the arrows. In each panel, the order of the lanes is:
lane 1, Ad2 fiber unboiled; lane 2, Ad5 fiber unboiled; lane 3, Ad7 fiber unboiled; lane
4, Ad2 fiber boiled; lane 5, Ad5 fiber boiled; lane 6, Ad7 fiber boiled.
13. Trimers generally run at a molecular mass approx three times larger than that of
monomers (see Fig. 3) (see Notes 11 and 12).
Adenovirus
Fiber Protein
257
3 Prehybrtdize the blot for 30 mm at room temperature with 5% nonfat dry milk m
PBS (BLOTTO hybrtdrzatton solution)
4 Dilute the RL-2 antibody (I* lOOO-1:2000 for a typical ascites fluid preparation of
enzyme) mto the BLOTTO hybridization solution Incubate at room temperature
for l-2 h.
5 Remove the hybridization solutron and wash three times wrth fresh BLOTTO
hybrtdization solution.
6 Add goat antrmouse Ig antibody ConJugated to alkaline phosphatase (Frsher
Brotech) and incubate for 1 h at room temperature (see Note 10)
7. Wash three times with PBS or with carbonate buffer (pH 9.8, 5 mm each wash)
8 Develop the color with an appropriate substrate for alkaline phosphatase, accordmg to the manufacturers mstructions.
of
label added to fiber could be significantly lower, smce many of the possible
contaminating proteins may also contain O-GlcNAc that ts more accessible to
galactosyltransferase than are the O-GlcNAc moieties on fiber
3 4 2.1. PREGALACTOSYLATION OF THE GALACTOSYLTRANSFERASE ENZYME
1. Prior to the reaction, the enzyme itself must be pretreated with unlabeled UDPgalactose, because the enzyme itself contains significant quantities of O-GlcNAc
(40-43). 1.5 mg of bovine mtlk 4/3-galactosyltransferase is added to a I-mL
solution that contains 200 mA4 sodmm cacodylate (pH 6.8) and 5 mM MnC12.
As an alternative, 50 mM HEPES (pH 7 3-7.5) may be substituted for the
cacodylate buffer
2. Add 2 m/k2 unlabeled UDP-galactose, 3.5 pL of a 1:50 dilution of p-mercaptoethanol m H20, and 10 pL aprotmin. The reaction IS carried out for 30 mm
on ice, then 60 mm at 37C
3 Add 5.66 mL of saturated ammonium sulfate solution at 4C (85% final concentration after dtlution) and incubate on ice for 30 min
4 Spin at 10,OOOg for 15 mm m a refrigerated centrifuge. Typically, this centrrfugation is performed m an SS-34 rotor (Sorvall)
5. Remove and save the supernatant
6. Resuspend the pellet m 5 mL of 85% ammomum sulfate prechtlled to 4C. Incubate for 30 min on ice
7 Spm at 10,OOOg for 15 mm m a refrigerated centrifuge. Typically, this centrrfugation IS performed m an SS-34 rotor (Sorvall)
8 Remove and save the supernatant.
9. Resuspend the pellet m 1 mL of 25 mM HEPES (pH 7.4), 2.5 mA4 MnCl,, and
50% glycerol The approximate enzyme activity is 25 mU/mL.
252
253
254
References
1. Svensson, U , Oersson, R., and Ever@ E. (1982) Virus-receptor Interaction m the
adenovirus system I Identification of vlrion attachment proteins of the HeLa cell
plasma membrane. J. Vwol. 38, 70-8 1.
2. Devaux, C., Caillet-Boudm, M.-L , Jacrot, B , and Boulanger, P (1987) Crystallization, enzymatic cleavage, and the polarity of the adenovlrus type 2 fiber
Vzrology 161, 12 l-l 28
3. Greber, U F., Willetts, M , Webster, P., and Helemus, A. (1993) Stepwlse dlsmantlmg of adenovirus 2 during entry into cells. Cell 75477-486
4. Louis, N., Fender, P., Barge, A., Kltts, P , and Chroboczek, J (1994) Cell-binding
domain of adenovlrus serotype 2 fiber. J Vwol 68,41044106
5 Bal, M., Harfe, B , and Frelmuth, P. (1993) Mutations that alter an Arg-GlyAsp (RGD) sequence m the adenovirus type 2 penton-base protein abolish rts
cell-rounding activity and delay virus reproduction in flat cells. J Viral 67,
5 198-5205.
6 Wickham, T. J , Mathias, P., Cheresh, D. A., and Nemerow, G R. (1993) Integrin
a@3 and a$5 promote adenovlrns mtemahzatlon but not virus attachment Cell
73,309-3 19
Adenovws
Fiber Protein
255
256
17,
Adenovirus
fber
Protein
257
42. Roquemore, E P , Chou, T. -Y , and Hart, G W. (1994) Detectlon of O-linked Nacetylglucosamme (0-GlcNAc) on cytoplasmlc and nuclear proteins Methods
Enzymol. 230,443-4&I.
21
Large-Scale Purification
and Crystallization
of Adenovirus
John J. Rux, Donatelia
Hexon
1. Introduction
Adenovirus, shortly after its discovery in 1953 (I), was one of the first biological entities to be imaged using the electron microscope (EM) (2). Since
then, It has been under continuous scrutiny by structural btologtsts
(reviewed m ref. 3). The characteristic shape of the adenovu-us virion, with
its projecting fibers, made it a fascinating object for early EM studies, which
focused on how its >lO structural proteins are organized to form the vnton.
The size of the vu-ion, with a diameter of 9 14 A at the fivefold verttces (4) and
a mass of over 150 x 1O6Dalton, presents a formidable challenge for X-ray
crystallographic studies.
An alternative approach to determining the virion structure was facilitated
by the property that cells infected with adenovu-us contain a lo- to 1OO-fold
excess of most structural proteins in soluble form over those incorporated into
virions (s), which permits the isolation of these proteins for structural studies.
The major coat protein, hexon, was the first animal-virus protein to be crystallized (6), and its three-dimensional structure has since been determined m progressively increasing detail (7-9) As hexon represents >60% of the total
particle weight (IO), its initial low-resolution structure could be used to develop
a model for the adenovirus capsid (11). Further studres using cryo-electron
microscopy and image reconstruction (4) revealed the complete virion to 35 A
resolution. Subsequent work combined the crystallographic and EM images
and revealed details of the minor proteins (12).
In this chapter, we describe a protocol for the large-scale purification for
virions of adenovrrus type 2 (Ad2) and Ad5 and the soluble major coat protein,
hexon. The virus particles can be used as seed stock for further rounds of infecFrom
259
and Protocols
NJ
260
tion, for EM studies on the intact particle or its fragments (13) or m gene
therapy mvestlgatlons (14). The hexon may be used to produce crystals sultable for high-resolution X-ray crystallographic studres, as described here. An
extension of this protocol to other adenovn-us types depends on the ease of
viral growth and the quantity of soluble hexon found m the infected cell Several members of subgroup C (types 1,2, 5, and 6) have been crystallized from
soluble protein (15-17), but the fastidious subgroup F vu-uses (types 40 and
41), which grow only in 293 cells, yield little hexon (28). Structural studies on
other structural proteins such as the fiber (19), its receptor-bmdmg knob (20),
and the complete penton (22), which are present in low quantities m the infected
cell, are being faclhtated by expression systems. It may be difficult to extend
these to hexon, which has a complicated foldmg pathway requiring the presence of the adenovn-us-coded nonstructural 100-kDa protem (22).
The hexon purlficatlon scheme using amon-exchange chromatography has
been developed in our laboratory over several years and 1sbased upon a variety
of other pubhshed protocols (23-28), as well as our own experience. The protocol has been optimized to provide the large amount of protein required for
structural studies m as short a time as possible. Once preparations have been
made, the procedure requires approx 1 wk to obtain between 10-20 mg of
purified hexon (see Note 1).
2. Materials
The materials listed are those routmely used to perform the adenovirus
hexon purification protocol. Substltutlons could be made with equivalent
items as available.
2.1. Cells and Virus
Human Ad2 and Ad5 are routinely propagated m suspension cultures of
HeLa S3 cells as described; Ad2 (ATCC VR-846) and Ad5 (ATCC VR-5) and
HeLa S3 cells (ATCC CCL 2.2) are available from the American Type Culture
Collection (ATCC, Rockvllle, MD).
2.2. Stock Solutions
All stock-buffer solutions are filtered through 0.22-p
(Corning, Corning, NY, cat. no. 25960-33).
1 Jokhks
bottle-top filters
powder
261
11. Leupeptm (1 mL). 2 mg leupeptm (Sigma) m water, store at -20C Make freshly
for each preparation
12 Pepstatm (1 mL) 1 mg pepstatm (Sigma) m 1 mL (200 proof) ethanol; store at
-20C. Make freshly for each preparation.
13. PMSF (1 mL). 17 mg phenylmethylsulfonyl fluoride (Sigma) m 1 mL (200 proof)
ethanol, store at -20C Make freshly for each preparation. Cautzon. Both the
powder and solution are highly toxic, gloves are required
14. Trypan blue stain 0 4% (100 mL) (Glbco-BRL) Store at 25C.
15 Virus assay buffer. 20 mM sodium phosphate, pH 7.2 (Sigma), 0.5% SDS (BloRad, Hercules, CA)
16. DMSO* dimethylsulfoxlde (Sigma).
17 Freon: 1,l ,ZTnchloro-1,2,2-tnfluoroethane, Cl,FCCC& (Mallinckrodt, Phillipsburg, NJ)
18 High-vacuum grease (Fisher, Pittsburgh, PA)
19. Dlsmfectant cleaning solution. Amphyl (Spectrum Chemical, Houston, TX).
2.3. Equipment
1. Spinner flasks. 2 x 50 mL, 100 mL, 250 mL, 500 mL, 1 L, 3 L (Belco, Vmeland, NJ)
2. Conical-bottom centrifuge bottles: 4 x 750-mL (Belco)
3 37C COz-incubator, or warm room (see Note 4), two heavy-duty magnetic stir
plates (Bell Stir, Belco)
4. Lammar flow hood.
5 Hemacytometer with improved Neubauer ruling (Fisher); sterile-disposable I -mL
plpets, and microscope.
6. Eppendorf plpetteman pipets: lOOO-, loo-, and lo-@ tips.
7 Dlsposablesn I- and 5-mL syringes (Beckton and Dickinson, Rutherford, NJ),
50-mL tubes (Falcon, Los Angeles, CA); 0.45~pm syringe filters, Centrlprep PM
262
8.
9
10.
11.
12
3. Methods
3.1. Preparations
solution
solution remains clear. If the solution 1s cloudy or contains suspended partlculates, 1t is contaminated and must be discarded. The remaining solutions may
be prepared after the HeLa cell culture is underway. The density of the TrisCsCl solutions should be checked by measuring their refractive indices, and
adding either distilled/deionized water or CsCl to the solutions as necessary to
obtain the correct values.
3.1.2. HeLa Cell Culture
A starting culture of S3 HeLa cells must be obtained from either ATCC or
another laboratory. The cells are then maintained 1n a suspension culture as
described below (see Notes 4-9). All work with cells is done m the laminar
flow hood to maintain sterile conditions (see Note 6). If the cell culture
becomes contaminated, it must be discarded and another culture started.
1. Thoroughly clean and autoclave the spinner flasks, letting them cool to room
temperature.
2 If a starter culture IS available 1n suspension, skip steps 3-6
3 Warm a 500-mL bottle of complete media 1n a 37C water bath
4 Thaw the frozen cells 1n a 37C water bath for 1 min.
5. Within the lamlnar flow hood, transfer cells to a sterile 15-mL tube and add
warm complete media dropwise over a period of 10 mln, slowly bring the
volume up to 15 mL
263
6 Sediment the cells at 1OOOgfor 10 min, decant off media, add fresh complete
9.
10
11
12
media, and resuspend cells by gently and repeatedly drawing the solution mto a
sterile 25-mL plpet and releasmg. Sufficient volume IS needed to cover the stirbar m the spinner flask (e.g., 25-mL culture for a 50-mL spinner flask, or 40 mL
for a lOO-mL spinner flask) to keep the cells growing well
Remove a l.O-mL fraction from the culture and count the number of cells (see
Notes 7-9) AdJust the cell concentration to 2-5 x lo5 cells/ml, cells can be
sedimented at 1OOOgfor 10 min and excess media decanted If necessary.
Log-phase HeLa cells have a doubling time of 24 h The suspension culture must
be split regularly to mamtam a cell concentratton of 2-10 x lo5 cells/ml. Cells
are split every 2-3 d by discarding a portion of the culture and replacing the
discarded volume with complete media.
It is easier and less expensive to maintain a small volume of cells in suspension,
and to scale up only when necessary.
To scale up the cell-culture volume, do not split the culture, but increase its
volume with fresh complete media to bring the cell concentration to 2-5 x IO5
cells/ml.
Begin to scale up the volume of the suspension culture from approx 100 mL to
2 5 L 5-6 d before the cells are to be mfected
For long-term storage, the cells can be concentrated to 10 cells/ml, frozen overnight at -70C m a mixture of 50% Jokllk solution, 40% horse serum, 10%
DMSO, then stored under liquid nitrogen.
3.1.4 Chromatography
Column chromatography is conveniently performed using a Pharmacla
FPLC within a chromatography refrigerator. The chromatography solvents,
columns, and samples are kept at 4C. The DEAE column must first be packed
Then, both the DEAE and Mono-Q columns must be equilibrated with column
buffer by flushing with five column volumes of 10 mM BTP (pH 7.0) before
use. If an automated system like the FPLC is to be used, methods for sample
loading, column flushing, and elution should be programmed before starting
(see Note 10). These methods can be quickly modified (e.g., based on sample
volume) as necessary Just before use.
264
3.2.,
steps 2-3).
2. Place contaminated glass pipets into 250-mL cylinder containing disinfectant
265
3. Decant media mto a large spinner flask, leaving enough media to cover 80% of
the conical bottom of the four 750-mL centrifuge bottles,
4. Mix the cells and media remaining in the bottles by slowly swu-lmg the solutton.
Gently draw the cells up and down with a 25-mL pipet to fully resuspend the
cells without breaking them.
5 Transfer the cell suspension to 50-mL Falcon tubes on Ice (three tubes with
approx 30 ml/tube).
6. Rinse bottles sequentially with 20 mL media from the 3-L spinner flask, and
divide the 20-mL rmse mto the three Falcon tubes.
7. Sediment the cells at 1OOOgfor 10 min.
8. Aspirate the supernatant mto a flask containing dtsmfectant.
9 Resuspend the cells mto cold PBS with a 25-mL pipet. Use 10 mL PBS per 1 L of
initial culture volume (e.g., 25 mL PBS for 2.5-L culture).
10 Sediment the cells at 1OOOgfor 10 mm at 4C.
11 Aspirate the supernatant into a flask containing disinfectant
12 Resuspend the cells into cold 20 n-uVTris-HCl + 0 5 MNaCl with a 25-mL ptpet
Use 10 mL Trts-NaCl/l L of mtttal culture volume.
13 Add protease mhibitors. 50 pL leupeptm, 100 pL pepstatin, 100 pL PMSF, per
100 mL of cell suspension
14 Lyse cells by repeatmg five freeze-thaw cycles.
a Freeze cell suspension m an ethanol dry-ice bath for 45 mm
b Thaw cells in lOOO-mL beaker of warm water, mix the cell suspension by
swirling the Falcon tubes m the warm water bath
c Forcibly pipet the cell suspension up and down 20X with a 25-mL pipet for
each tube to help break up cells and debris. If the color of the solution begins
to turn brown, add more Tris-NaCl buffer to each tube to keep the solution
basic. After freezmg cells for the fifth time, the solutton can be stored frozen
at -20C overnight
15. Sediment cell debris.
a Finish the fifth freeze-thaw cycle.
b. Pellet the DNA by centrifugatton at 3000g for 10 mm
16. Decant supernatant mto three new Falcon tubes, store on Ice.
17. Resuspend DNA pellets m 10 mL Tris-NaCl buffer.
18. Pellet DNA by centrtfugation at 3000g for 10 mm.
19 Combme supernatants, mtx them by pouring solutton from tube to tube, and
divide solutton into four tubes of approx 15 mL each.
20. Add 15 mL Freon to each tube, cap tubes tightly, and seal them with paratilm.
2 1 Shake tubes vigorously by hand for 5 mm each.
22. Separate the organic from the aqueous phase by centrtfugation at 2000g for
10 mm.
23. Pipet off the top layer (aqueous phase) and place it m two new Falcon tubes
24. Re-extract the organic phase with an additional 2 mL Tris-NaCl buffer, and combine with the aqueous layer from the previous extraction
25 Cool the ultracentrifuge and the SW-28 rotor to 5C.
266
267
Fast Flow
(salt gradient)
SDS-PAGE
(silver stained)
20 24 28 32 36 40
44 48 52 56 60 64
Fig. 1. First column: Purification of adenovirus hexon by DEAE-sepharose fastflow chromatography and analysis by SDS-PAGE. The chromatogram is a tracing of
A 280nm and the gradient is a tracing of specific conductivity. Fractions spanning the
hexon peak are labeled and are analyzed by silver-stained SDS-PAGE. The position
on the gels that corresponds to the hexon band is labeled (Hx).
c. Puncture the tube through the grease with an 18-gage needle attached to a
3-mL syringe with the needle bevel facing up (toward the virus band).
d. Raise the tip of the 1S-gage needle into the virus band by gently lowering the
barrel of the syringe and carefully draw out the virus band without disturbing
the gradient.
11. Pool the virus-containing fractions.
12. Add glycerol to a final concentration of 20% (v/v).
13. Mix solution, place lOO-uL aliquots into labeled cryotubes, and store them in
a -20C freezer.
3.5. Purification
of Hexon
The purification
consists of three separate column chromatography
steps.
The first step uses a DEAE-Sepharose
fast flow anion-exchange column that
separates the hexon from crude cell lysate (see Note 12). The hexon is then
further purified by anion-exchange chromatography
with Mono-Q columns.
3.5.1. DEAE-Sepharose
Fast-Flow Chromatography
268
SDS-PAGE
Mono Q (lo/lo)
18
(silver stained)
(salt gradient)
16 17
19 20 30
A /II\\ I
16 17 18 19 20 30
chro-
4. Dilute the sample 1.1 with column buffer to reduce the sample viscosity
5 Prepare solvents
A* 10 m/V BTP (pH 7.0).
B 10 & BTP (pH 7.0) + 1.O MNaCI.
6. If an automated system like the FPLC IS to be used, warm up the absorbance and
conductlvlty detectors, then program a method to equlhbrate the DEAESepharose fast-flow column (30 x 1.6 cm, 60 mL) with solvent A at 1 0 mL/mm
until the specific conductance has stabilized
7 Load the diluted sample at 1.O mL/mm
8 Step the gradient to 10% B and flush the column with two column volumes (120 mL)
at 1 0 mL/mm Save the solvent flow-through m case the column falls to bmd hexon
9. After proteins that do not bmd to the column have eluted, begin collectmg fractions of 4 ml/tube, and start a gradient from 10% to 100% B over 400 mL
10. Step the gradient back to 0% B and wash for 5 column volumes to re-equilibrate
the column with solvent A
HR lO/lO column.
The protocol
of adenovlrus
is as follows.
hexon on
2
3
5
6
7
8.
9
10
11
269
Etther pool and dialyze the fractions contaming hexon, as described above (Subheading 3.4., step 6) or concentrate the protein and exchange its buffer as described below.
a. Place up to 15 mL of the fractions into a Centriprep PM 10; add the fractions
contammg the least amount of hexon first.
b Spm at 3000g for 30 mm at 4C
c Collect the filtrate, add more fractions, repeat the concentration, and sample
addition cycle until the sample volume is less than 15 mL
Dilute the sample 1.1 with 10 mM BTP (pH 7.0) to reduce Its tome strength
and viscosity.
Prepare solvents:
A. lOmMBTP(pH70).
B: 10 mA4BTP (pH 7.0) + 1 OMNaCl
If an automated system like the FPLC is to be used, program a method to equilibrate the Mono-Q column (10 x 1.O cm, 8 mL) with solvent A at 2.0 mL/min until
the specific conductance has stabilized.
Load the diluted sample at 2 0 mL/min
Flush the column with 30 mL solvent A at 2.0 mL/mm Save the solvent flowthrough m case the column fails to bmd hexon
Step the gradient to 25% B; flush the column with 30 mL at 2 0 mL/mm
Begin to collect fractions of4 ml/tube, and start,agradient from 25 to 40% B over 50 mL
Hold the gradient at 40% B for 12 min (24 mL), step to 100% B, and flush column with 8 mL
Step the gradient back to 0% B, and wash with 5 column volumes to re-equihbrate the column with solvent A.
Assay the fractions by SDS-PAGE as before (Subheading 3.5.2.).
After anion-exchange
chromatography
with the Mono-Q (1 O/l 0) column,
the hexon-containing
fractions may still be contaminated
with significant
amounts of other proteins, as judged by SDS-PAGE analysis. If further punfi-
270
Mono Q (5/5)
SDS-PAGE
(salt gradient)
(silver stained)
16 17 18 19 20 21
16 17 18 192021
Fig. 3. Third column: purification of adenovirus hexon by Mono-Q (5/5) chromatography and analysis by SDS-PAGE. Labeled corresponding to Fig. 1.
5. Dilute the concentrated sample with storage buffer and concentrate again, repeat
this process until the sample has been exchanged 99% or more into the hexonstorage buffer.
3.6. Crystallization
of Hexon
After concentration and buffer exchange, the freshly purified hexon is used
immediately for crystallization trials. Hexon is crystallized (Fig. 4) using the
vapor-diffusion hanging drop method (30) (see Note 15). A drop of protein
solution is suspended over a reservoir of precipitation solution and allowed to
equilibrate. The gradual increase in precipitant concentration within the drop
under the appropriate conditions causescrystal formation. The steps for hexon
crystallization are as follows:
1. Heat a small amount of high-vacuum grease in a 25-mL beaker on a hot plate
until melted.
2. Place 1.O mL of 1.O A4 sodium citrate (pH 3.20) in the well of a 24-well tissueculture tray (see Note 3).
3. Place a thin layer of melted high-vacuum grease around the rim of the well.
4. Place a 5 $ drop of 10 mg/niL hexon solution on a 22-mm diameter siliconized cover slip.
5. Use a 20 pL pipetman to mix 5 clr, precipitation buffer from the reservoir with
the 5 pL hexon drop. Take care not to produce bubbles when mixing the solutions.
Fig. 4. Adenovirus type 5 hexon crystal (approx 0.5 mm) grown in a IO-pL hanging drop containing 5 pL (10.6 mg/mL) hexon and 5 uL of the 1 mL (1 .OM sodium
citrate pH 3.2) reservoir solution.
6. Place the cover slip over the well with forceps; gently push down on the rim of
the cover slip to ensure a complete seal.
7. Repeat steps 2-6 for each crystallization trial.
8. Store the crystallization tray at 25C where it will not be exposed to large temperature fluctuations, vibrations, or other disturbances that could upset the equilibrium of the drops (see Note 16).
4. Notes
1. Time table (the procedure should be performed as quickly as possible):
Preparation
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Subheading
Subheading
3.1.
3.2.
(free)
Subheading
Subheading
Subheading
Subheading
Subheading
2. Although not always feasible, it is best to use freshly prepared media for cell
culture (31). A key indicator of expired media is that the doubling time for the
cells will increase from 24 h to 2 or more days.
3. Hexon crystallization is highly sensitive to solution pH. It can be difficult to
consistently prepare the crystallization buffer with precisely the correct pH for
crystal growth. To ensure that a suitable crystallization solution is available, pre-
272
4.
7.
8.
9.
10
11
12.
273
13, Samples to be loaded onto the FPLC must not contain particulates that could clog
its valves, lines, and columns. Particulates may be removed either by filtration
with a 0 45-w membrane, or by sedimentation Crude hexon solutions can be
difficult to filter, so sedimentation IS the preferred method.
14. The protocol for the Mono-Q (5/5) column is analogous to that described for the
Mono-Q (lo/IO) column The following modifications should be made Use 1 mL/min
flow rate throughout. Change gradient profile after loading sample to. step gradient
to 25% B, flush with 40 mL, start gradient from 25 to 62.5% B over 125 mL, step
gradient to 100% B and flush column with 8 mL; step gradient back to 0% B, and
wash with 5 column volumes to re-equilibrate the column with solvent A
15 Hexon crystals have also been grown in capillary tubes (7), and from bulk solution as a final purrfication step (26).
16. Crystal growth should be apparent within hours of setup time. The crystals reach
0.3-O 5 mm maximum diameter in 3-5 d and grow no further after 2 wk. For
optimal results, avoid handling the crystallization trays for the first 3-5 d after
they have been set up.
Acknowledgments
We thank the many ptoneers who enabled our work by developing adenovn-us protocols over the years. In our own laboratory, Markus Grutter, Jantce
L. White, Jan van Oostrum, Paula R. Kuser, and Susan L. Pichla all have made
contributions.
Robert Rtcciardi, Dental School, and Krishna J. Fisher, Vector
Core Facility of the Institute for Human Gene Therapy, at the University of
Pennsylvania have been generous m providing us with adenovtrus. We are also
indebted to Loutse Showe for helpful dtscusstons regarding HeLa cell culture.
This work has been supported by the National Institute of Allergy and Infectious Diseases (AI- 17270), the National Science Foundation (MCB 95-07 102),
and by the Wistar Cancer Center (CA 108 15).
References
1. Rowe, W P , Huebner, R. J., Gillmore, L K., Parrott, R H , and Ward, T G
(1953) Isolation of a cytopathogemc agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc Sot Exp Blol Med 84,570-573
2. Horne, R. W., Brenner, S., Waterson, A. P., and Wildy, P. (1959) The icosahedral
form of an adenovirus J A401 Bzol 1,84-86
3 Burnett, R M (1996) The structure of adenovuus, m Structural Bzology of Vwuses
(Chm, W., Burnett, R. M , and Garcea, R., eds.), Oxford University Press, New
York, pp. 209-238.
4 Stewart, P L , Burnett, R M , Cyrklaff, M., and Fuller, S. D. (1991) Image
reconstruction reveals the complex molecular orgamzation of adenovirus. Cell
67,145-154
5
White, D 0 , Scharff, M. D., and Maizel, J. V., Jr. (1969) The polypeptides of
adenovirus III Synthesis in infected cells Vzrology 38, 395-406
274
10
11.
12
13
14
of an
adenovirus protein (the hexon) Nature 219,946,947
Burnett, R M , Grutter, M. G., and White, J L (1985) The structure of the adenovnus capsid. I An envelope model of hexon at 6 A resolution J A401 Brol 185,
105-123.
Roberts, M M., White, J. L., Grutter, M G , and Burnett, R M (1986) Threedimensional structure of the adenovnus maJor coat protein hexon. Sczence 232,
1148-1151.
Athappilly, F K., Murah, R., Rux, J J , Cal, Z , and Burnett, R. M (1994) The
refined crystal structure of hexon, the maJor coat protein of adenovuus type 2, at
2 9 A resolution. J Mol BEOI 242,430-455
van Oostrum, J. and Burnett, R M (1985) Molecular composition of the adenovlrus type 2 virion. J Vwol 56,43!%448
Burnett, R. M (1985) The structure of the adenovtrus capsid II The packing
symmetry of hexon and its implications for viral architecture. J Mol. Bzol 185,
125-143
Stewart, P L., Fuller, S D., and Burnett, R M (1993) Difference imaging of
adenovn-us. Bridging the resolution gap between X-ray crystallography and electron microscopy. EMBO J 12,2589-2599
Furcmitti, P S , van Oostrum, J , and Burnett, R. M. (1989) Adenovnus polypeptide IX revealed as capsid cement by dtfference images from electron microscopy
and crystallography EMBO J 8, 3563-3570.
Kozarsky, K. F. and Wilson, J. M. (1993) Gene therapy. adenovnus vectors Curr
Open Genet Dev 3,499-503.
of the receptor-binding domam of adenovnus type 5 fiber protein at 1.7 A resolution Structure 2, 1259-1270
2 1. Schoehn, G , Fender, P , Chroboczek, J , and Hewat, E A. ( 1996) Adenovuus 3
penton dodecahedron exhibits structural changes of the base on fibre bmdmg
EMBO J. 15,68414846
22 Cepko, C. L. and Sharp, P. A. (1982) Assembly of adenovnus maJor capsid pro-
275
22
Adenovirus
Protease
Joseph M. Weber
1. Introduction
Adenoviruses encode a cysteme endopeptldase synthesized late m virus
infection which is essential for virlon maturation and infectivity (I,2) The
enzyme 1sencapsldated (approx 20 molecules per vlnon) and may also have a
role durmg decapsldation (3-5). Although there are approx 100 adenovirus
serotypes known to infect vertebrates, so far only the human adenovirus
type 2 (Ad2) enzyme has been studied. The recombinant protein expressed
in Escherzchia colz and insect cells has been purified and characterized
(3,6-a). The enzyme is a 204-residue monomer of 24,838 Dalton with a pI
of 10.59 and optimal activity at 45C and pH 8.0. The recombinant enzyme
is stimulated by an 1l-amino acid cleavage fragment from viral protein
pre-VI. GVQSLKRRRCF. The peptide 1s presumed to regulate enzyme
activity in vivo during virus infection. It is bound to Cl04 on the enzyme
via a dlsulphide bridge (9). Mutational analysis and X-ray chrystallography
identified the active-site triad as H54-C122-E71 (S-11). The substrate
specificity of the enzyme 1s(M,I,L)XGG-X
or (M,I,L)XGX-G
(12). Alkylating agents and E64 inhibit the protease and virus infection as expected
(13). Specific inhibitors are not yet available. To date, 17 protease genes have
been sequenced m different virus serotypes. The translated ammo acrd
sequences range from 20 1 to 2 14 residues and show both variable and highly
conserved regions (1,11).
2. Materials
1. Buffer A: 10 mJ4 Tris-HCl, pH 8 5, 1 mM EDTA, 5 mM mercaptoethanol,
10% glycerol
2 Buffer B* 10mMphosphatebuffer, pH 6 7, 1WEDTA, 5 mMmercaptoethano1,
10% glycerol.
From
Melhods in Molecular
Medrone,
Vol 21 Adenovrrus
Methods
E&ted by W S M Wold 0 Humana Press Inc , Totowa,
277
and
NJ
Protocols
Weber
278
3. Methods
3.2. Chromatographic
Purification of Protease
Adenovirus
Protease
279
4. Notes
1 The protease has been expressed as a fusion protein with N-terminal tags of protein A or hexahistidme to facilitate purification. These tags did not appear to
interfere with enzyme activity.
2 Preparations of recombinant protease require the addition of a peptlde to acquire
full-enzyme actlvlty (4,8,15). The enzyme should be incubated (15 min at 37C)
280
Weber
References
1. Weber, J. M. (1995) The adenovtrus endopeptrdase and Its role m vu-us mfectron,
m Molecular Repertowe ofAdenovzruses (Doerfler, W. and Petra Bohm, P , eds ),
Curr Topics Mwroblol Immunol. 1994 pp. 227-235
2 Weber, J. M and Trhanyt, K. (1994) Adenovirus endopeptrdases Methods
Enzymol 244D, 595S604.
3. Anderson, C. W (1990) The protemase polypepttde of adenovnus serotype 2 vtrtons. Vwology 177,259-272.
4. Cotten, M. and Weber, J M. (1995) The adenovnus protease IS required for vnus
entry mto host cells. Vwology 213,494-502
5 Greber, U. F., Webster, P , Weber, J., and Helenms, A. (1996) The role of the
adenovnus protease m vn-us entry mto cells EMBO J 15, 1766-l 777.
6 Houde, A. and Weber, J M (1990) Adenovnus protemases compartson of ammo
acid sequences and expression of the cloned cDNA m Escherlchla COIL Gene 88,
269273
7. Tlhanyl, K , Bourbonmere, M., Houde, A, Rancourt, C , and Weber, J M (1993)
Isolation and properties of the adenovtrus type 2 protemase J Blol Chem 268,
1780-1785
8 Webster, A., Hay, R. T , and Kemp, G (1993) The adenovuus protease IS actlvated by a vn-us-coded disulphide-linked peptide. Cell 72,97-104
9 Ding, J , McGrath, W. J , Sweet, R. M , and Mangel, W. F (1996) Crystal structure of the human adenovnus protemase with its 11 ammo acid cofactor. EMBO J
15,1778-1783.
10 Grterson, A W., Nicholson, R., Talbot, P , Webster, A., and Kemp, G. (1994) The
protease of adenovn-us serotype 2 requires cysteme residues for both actlvatton
and catalysis. J. Gen. Vwol. 75, 2761-2764.
11. Rancourt, C , Tthanyt, K., Bourbonnibre, M , and Weber, J M. (1994) Identrficanon of active-site residues of the adenovnus endopeptldase Proc Nat1 Acad
SCL USA 91,844-847
12 Webster, A., Russell, W. C , and Kemp, G. D (1989) Characterrzatlon of the
adenovnus protemase, substrate specificity J Gen Vzrol 70, 32 15-3223
13 Strcar, S., Keyvam-Amineh, H , and Weber, J M. (1996) Inhrbmon of adenovn-us
infection with protease inhtbttors. Antlvrral Res 30, 147-153
14 Mangel, W. F , McGrath, W. J., Toledo, D L., and Anderson, C W (1993) Viral
DNA and a viral pepttde can act as cofactors of adenovnus vtrton protemase
activity. Nature 361,274,275
Adenovirus Protease
281
15. Dloun, M., Geoghegan, K. F , and Weber, J. M. (1995) Functlonal charactenzation of the adenovirus proteinase usmg fluorogemc substrates. Protew Pep&e
Lett 6,363-370.
16. Mangel, W. F., Toledo, D L., Brown, M. T., Martin, J H., and McGrath, W. J
(1996) Characterization of three components of human Adenovu-us proteinase
activity in wtro. J Blol Chem 271,536543.
23
Methods for Growth and Purification
of Enteric Adenovirus Type 40
Vivien Mautner
1. Introduction
The enteric adenoviruses of subgroup F (Ad40 and Ad41) pose some special
problems of cultivation, as they cannot be readily passaged in many of the cell
types used to propagate the more commonly used subgroup C serotypes (Ad2
and Ad5), and there is no standard plaque assay: A full dtscusston of the observations from many laboratories is mcluded in a recent revtew (I). For the purposes of this chapter, I will describe the methods developed m my laboratory
to passage Ad40 m 293 and KB16 cells, to assay the virus by a variety of
methods, to purify the vnus by cesium chloride density gradient centrifugation, and to obtain viral DNA. Most of the methods have been derived from
those developed for Ad5, but with constraints imposed by the more fasttdtous
nature of the entertc adenovnuses.
Ad40 stram Dugan (2) is available from the American Type Culture Collection (ATCC, Rockville, MD). In my laboratory, the virus was passaged nme
times m KB 16 cells (31, using a 1: 10 dilution of the total yield at each step to
provide a p9 stock with a titer by fluorescent focus assay of 6 x lo6 FFU/mL
and a particle count by electron microscopy of 2 x 10 particles/ml. This
virus was used for all experimental work, and a virus stock grown from this
source was used to make DNA for cloning and sequencing of the complete
Ad40 genome (4) (Genbank accession number L19443). The virion DNA was
shotgun cloned into bacteriophage M 13mp 19 and sequence data generated randomly; from the proportion of nonviral sequences cloned and sequenced, it
was possible to estimate that the virion-derived DNA was 98.6% pure.
In order to generate substantial amounts of Ad40, we attempted to passage
vn-us seed stocks at a very low multtplicity, as is the procedure for Ad5, but tt
From
Methods
Edlted
m Molecular
by
Medune,
W S M Wold
Vol 21 Adenowrus
0 Humana
283
Press
Methods
Inc , Totowa,
and Protocols
NJ
284
Mauiner
became apparent that seed stocks diluted beyond 1:30 failed to propagate. We
therefore routinely maintain virus stocks by serial passage at 1: 10 dilutions,
and check the yield of virus at each step by comparing levels of virion packaged DNA. It is also important to check the restriction profile for inadvertent
contamination
with other serotypes; if other adenovtruses are used in the
laboratory,
this is not a trivial consideration.
A comprehensive
survey of
the restriction profiles of the human adenovirus serotypes (Ad1 to Ad41) is
available (5).
Although tt has been reported that A549 cells will support plaque formation
of the Sapporo strain of Ad40 (61, the Dugan strain cannot be plaqued in 293
cells, so a fluorescent focus assay has been used to estimate the number of
infected cells and thence the titer of virus in a stock. For Ad5, the FFU-to-PFU
ratio is approx 10: 1, and the particle-to-PFU
ratio ranges from 10 to 100. For
Ad40 passaged in 293 or KB16 cells, we find a particle-to-infectivity
ratio in
the order of 103; and although the reason for this large difference is not established, a number of explanations have been suggested (I, 7,8)
2. Materials
1. Ad40 strain Dugan. ATCC VR-93 1 (2).
2. 293 cells. ATCC CRL- 1573 (human embryonic primary kidney cells transformed
with sheared Ad5 DNA, expressing E 1a+b) (9)
3. KB 16 cells: (KB epidermoid-carcmoma
continuous cell line (ATCC CCL-17)
further transformed with Ad2 E 1a+b DNA) (3).
4. Dulbeccos modified Eagles medium (DMEM) Gibco-BRL, Gaithersburg, MD
cat. no 04 1-O1965 M (without sodium pyruvate, with 4500 mg/L glucose) Store
at 4C
a Pen/strep solution Gibco-BRL cat no. 043-05 140 H (1000 m/mL pemclllm,
1000 pg/mL streptomycin). Ahquot and store at -20C.
b L-Glutamme: Glbco-BRL cat no. 043-05030 H (200 mM) Aliquot and store
at -20C
c. Serum (fetal calf): heat treat 56C for 3&45 mm. Aliquot and store at -20C
5 Cell-propagation
medium. DMEM (500 mL), Pen/strep solution (5 mL),
L-glutamine (10 mL), fetal calf serum (50 mL).
6. Infection medium* DMEM (500 mL), Pen/strep solution (5 mL), L-glutamme
(10 mL), fetal calf serum (2 5 mL).
Working stock should be stored at 4C and used within 10 d
7. Dulbeccos PBS (complete). NaCl(l37 mM), KC1 (2.7 mM), NaH2P04 (8.0 mM),
KH,PO, (2.4 m&Q, CaCl, (6.8 mM), MgCl, (4 9 mM)
For complete (PBS) use 8 parts solution A, 1 part solution B, and 1 part
solution C.
a. Solution A* NaCl(l0 g), KC1 (0.25 g), Na2HP04 (1 43 g), KH2P04 (0 41 g),
pH72per1LH20.
b. Solution B. CaC12*2H20 (1 g) per 1 L
8.
9.
10.
11
12.
13.
285
(0 5 mL), H,O to 100 mL. Use stertle reagents, store at room temperature.
14 Hut buffer
10 mM Trts-HCl,
pH 7 9, 10 mM EDTA, 0 6% (w/v)
SDS Filter-
3. Methods
3.1. Propagating
Cells
Mautner
286
every 7 d to maintain the stock. One 80% confluent flask yields approx 2 x 1O7
cells, which will provide 20 60-mm plates seeded at IO6 cells/plate. These
should reach 80% confluence u-r2 d. JCL316 cells are split 1.4 every 34 d; they
are less tolerant of a higher dilution.
1 Remove medium from the cells, add 5 mL prewarmed T/V, gently wash
2
3.
4.
5.
6.
cells untrl pH becomes acid. Dtscard T/V, add a further 5 mL and incubate at
37C until the cells detach (usually a maxtmum of 5 mm, do not leave longer
than necessary).
Bang the flask to detach all the cells, allow to dram to base of flask.
Add 20 mL prewarmed medium to the flask (serum m the medtum neutraltzes
the trypsm).
Disperse the cells by ptpetting up and down three times with a lo-mL ptpet
Seed flasks as requrred; e.g., 2.5 mL cell stock + 20 mL medium for a 1 10 spht
Regularly test all cell lines for mycoplasma, e.g., once every 2 mo Passage newly
recovered cells four times m anttblotrc-free medium before testing.
3.2. Preparing
1. Trypsmrze cells from a 90% confluent BO-cm* flask as described above and
of individual
carded accordingly.
1 Remove medium from subconfluent monolayers of 293 or KBa+b cells m
60-mm plates
2 Infect with virus diluted in TBS to gave approx 1 FFU/cell m 0.1 ml/plate (generally a 1: 10 dilution).
3 Adsorb 37C for 60-90 mm. Rock plates gently at 20-mm Intervals
4. Add 5 mL medium + 0 5% fetal calf serum.
5. Cells should take 5-7 d to show a good cytopathic effect (cpe) (Ad40 has less tendency
than Ad5 to produce a bunch of grapes effect, but mdrvtdual cells do round up)
6. When a good cpe 1s apparent, scrape cells into the medmm wrth a pastette or
teflon scraper, and prpet cells gently to drsperse clumps.
7 Pool cell suspensions and centrifuge at 9OOg for 10 mm at 4C
8. Dram pellets and resuspend in approx 0.25 mL TBS/60-mm plate
9. To release virus, freeze-thaw three times, I.e., freeze m dry me or hqutd nitrogen,
and thaw at 37C m water bath
Enter/c Adenowrus
Type 40
287
3.4. Preparation
1
2.
3.
4.
5.
6.
in 80-cn? Flasks
1.
2.
3
4
5
6.
7.
Make large quantities ofvuus stock (e.g., from thirty 60-mm plates or ten SO-n&. flasks)
Add l/l00 volume n-butanol and incubate 4C for 60 mm
Centrifuge at 9008 for 10 mm to remove cell debris
Remove supernatant containing virus
Layer on a CsCl/glycerol gradient (see below).
Centrifuge at 80,OOOg for 90 min at 4C. Do not use brake to stop.
Virus band should be visible to naked eye as the lowest opalescent band To see
more clearly, place m front of a black background and shine a light down the tube
from directly above There may be one or more bands above the vuus band, these
consist of empty or incomplete virus particles.
8. Collect the virus band by piercing the tube with a hypodermic needle below the
band, and collectmg drops into sterile 1-mL cryotubes. The fraction containmg
the virus band will be opalescent.
9. Remove CsCl either by dialysis against 10 mA4 Tris-HCI, 1 mM EDTA, pH 8 0,
or on a G50 Sephadex desalting column.
10. Mix vn-us with an equal volume of sterile 80% glycerol and store at -70C.
Banding
of Virus
288
Mauiner
CsCl can be removed by dialysis or desalting: the vu-uswill be diluted sometwoto fivefold, and there is a risk that the vu-uswtll aggregateduring theseprocedures.
The method of choice for desalting ~111be determined by the purpose for which the
material ISintended. For parttcle countsand other assaysof vuus concentratton,vuus
can be desalted but glycerol should not be added. Vnus intended for subsequent
infection can be stored at -70C with addition of glycerol after desalting.
1 Desalt on a G50 Sephadex column pre-equilibrated with TBS; or
2 Dialyze vs TBS OY60 mm Tris-HCl, pH 7.5,lO mA4 EDTA, 2% glycerol or vs
10 mM Trts-HCl, pH 7 6, 1 mM MgC12, 10% glycerol, 0 5% n-butanol (II),
and/or
3 Mtx vnus with an equal volume of sterile 80% glycerol and store at -70C.
3.9. Preparation
1.
2.
3
4.
(12)
Enter/c Adenowrus
2
3
4.
5
6.
Type 40
289
b If cells are detaching from plate, scrape cells mto medmm, centrifuge at 2000g
for 10 mm or at 15,000g for 1 min, resuspend cell pellet in 0.6 mL Hu-t buffer,
transfer to Eppendorf tube, and incubate for 10 mm at room temperature.
Add 0.15 mL 5 M NaCl, mix gently by inverting tube.
Incubate on ice overnight
Centrifuge at 15,000g for 30-45 min.
Remove supernatant (sometimes it is easier to remove pellet with a wooden
toothpick).
Precipitate DNA from supernatant using either of two methods
a. Add equal volume lsopropanol, incubate -20C overnight, centrifuge at
12,000g for 5 mm, and drain pellet.
b Add protemase K to final concentration 500 pg/mL, incubate 37C for 3 h,
extract once with phenol/chloroform, extract once with chloroform, EtOH
precipitate with 0 3 M sodium acetate, and wash pellet in 70% EtOH
For smaller samples (e.g., from Lmbro well), 35mm plate, add salmon
sperm DNA or tRNA as carrier for DNA precipltatlon.
3.12. Fluorescent
1
2
3.
4
5.
6.
Focus Assay
Mautner
290
7 Remove medium; wash twice with complete PBS Inspect cells to confirm morphology and adherence.
8 Fix with ice-cold 90% methanol x 4 mm
9. Wash twice with PBS Plates may now be stored at 4C with 1 mL PBS overlay
10. Add antibody at diluttons 150, 1.150, l-450 m PBS at 0 4 ml/plate As a control,
use preimmune serum where available. Additional control* Omit first antibody
11 Incubate at room temperature for 30 mm.
12. Save antiserum and store at 4C can be re-used at least twice
13. Wash plates twice m PBS.
14 Add 0 4 mL per plate fluorescem-conjugated swme anttrabbtt IgG (Nordic) and
rhodamme-conjugated BSA at appropriate dilutton; this needs to be determmed
emptrtcally. Swme anttrabbit serum can be re-used at least twice
15. Shake every 10 min at room temperature for 30 min.
16. Rinse twice in PBS Plates can be stored m dark at 4C wtth 1 mL PBS
17 Read plates with UV mtcroscope x 10 objective, x 10 eyepiece with 1 mm gratmg
graticule
18 Count total cell number under phase contrast opttcs, and number of fluorescing
cells m same field
3.73. Alternative
Enter/c Adenovirus
291
Type 40
11, Mount cover slips m 1 1 glycerol: PBSa mix or gelvatol, inverted onto a glass
slide. Samples mounted in PBS cannot be stored, whereas with gelvatol samples can
be stored for at least a week at room temperature. An antifade agent such as
Citrfluor may be added to the mountmg matertal.
12. View in inverted fluorescence microscope with an FITC filter at x20 magmficatron
13 Count total cell number under phase-contrast optics, and number of fluorescmg
cells m same field
(14-l
7)
Ad5, but Ad40 is constdered to be sufficiently similar that they remain valid. It
should be noted that there are discrepancies between the values obtained by the
various methods, and it IS recommended that for consistency, one method
should be chosen and adhered to.
3.14.1. Absorbance at Azeonrn
1. Using banded vnus, dilute 1: 10 into 0.5% SDS, 0.1X SSC 1 ODZbOof virus in 0 5%
SDS, 0.1X SSC = 0 28 mg/mL protern This is equal to 11 x 10 virus particles
2 Extract viral DNA and measure ODZbO. 1 OD260 = lOi virus particles/ml.
of Virology,
292
Mautner
4. Notes
1 Safety constderations for workmg with Ad40. Ad40 IS generally constdered
to be nonpathogenic to adults, and IS charactertsttcally associated wtth tnfantile gastroenteritis (2)
2 The vnus is classified as nontumortgenic, and in fact only partral transformatron
can be achieved in vrtro (181. However, gtven that the vuus IS infectious by the
oral route, tt is prudent to confine work with large amounts of purified virus to a
designated and contained area The extreme resistance of Ad40 to UV irradiation
has recently been reported (19) The actual level of contamment ~111be governed
by local regulattons. The use of gloves and an approprrate srde or back-fastening
lab coat IS recommended
Manipulations of vu-us infected material should be designed to muumtze the
generation of aerosols, if maternal 1s vigorously shaken or vortexed, the tube
should be briefly centrtfuged prtor to openmg Waste matertals should be
rendered noninfectrous prror to disposal* 1% Virkon or 0 5% SDS IS sufftctent to macttvate vtrus, but It 1s important to ensure that soltd waste 1s etther
autoclaved or exposed to disinfectant for a sufftcrent length of ttme (at least
30 mm) before discardmg
The use of glassware and hypodernnc needles should be kept to a munmum; any
skm contact or needle-suck mJury should be thoroughly flushed with runnmg water
Vrrkon is suitable for external skin apphcatlon A drip tray should be used to contam spillages, which can be absorbed onto paper tissue soaked m dlsmfectant
Acknowledgments
This chapter IS dedicated to the memory of Hello Pereira, m whose laboratory I learned the fundamentals of adenovtrology. I am indebted to Gate Brown,
Nancy Mackay, and Angela Rinaldi, who helped to develop many of the methods described here, and whose expert technical assistance over many years is
much appreciated.
References
1 Mautner, V., Stemthorsdottir, V., and Bailey, A. (1995) The entertc adenovuuses,
in The Molecular Repertoire ofAdenovwuses (Doerfler W and Boehm P., eds ),
Curr Topzcs Mlcroblol Immunol 199/III, Springer Verlag, pp. 229-282
2 de Jong, J. C , Wigand, R , Krdd, A. H., Wadell, G., Kapsenberg, J. G ,
Muzerre, C. J., Wermenbol, A G., and Ftrtzlaff, R. G. (1983) Candtdate
adenovtruses 40 and 41. fastidious adenovnuses from human infant stool J
Med Vzrol 11, 215-231
3. Babiss,L. E., Young, C. S. H., Fisher, P. B., and Ginsberg, H S. (1983) Expression of adenovirus E 1A and E 1B gene products and the Escherzchzaco11XGPRT
gene in KB cells J Vu-01 46,454465.
4. Davtson, A. J., Telford, E. A. R., Watson, M. S., McBride, K., and Mautner, V
(1993)The DNA sequence of adenovnus type 40. J Mol B~ol 234, 1308-l 3 16
293
5. Adrian, T , Wadell, G., Hlerholzer, J. C., and Wigand, R. (1986) DNA restriction
analysis of adenovnus prototypes 1 to 41. Arch Vzrol. 91,277-290.
6. Hashimoto, S , Sakakibara, N., Kumai, H , Nakai, M , Sakuma, S , Chiba, S , and
FuJinaga, K (1991) Fastidious adenovirus type 40 can propagate efficiently and
produce plaques on a human cell line, A549, derived from lung carcmoma. J.
Vu-01 65, 2429-2435
24
Transfection Complexes Generated
with Adenovirus
and Polyethylenimine-Condensed
DNA
Matt Cotten, Mediyha Saltik, and Adam Baker
I. Introduction
Many types of vtral infection produce a transient permeabilization of the
Infected cell (see refs. I and 2; reviewed in refs. 3 and 4). A large tmprovement m transfection efficiency was obtained when this phenomenon was
applied to receptor-mediated gene delivery (5). A number of permeabiltzing
agents have now been used to enhance gene transfer, but adenovirus is by far
the most potent (6,7). The augmentation of DNA delivery by adenovirus particles is primarily due to enhancement of cytoplasmtc entry of DNA; however,
viral components may also serve additional functions that promote transfection, such as generating alterations in the cytoskeleton of the transfected cell
(81 or m promotmg nuclear entry (9,10). The most efficient varrations of this
method employ some form of linkage between the adenovirus and the plasmrd
DNA, thus ensuring that each viral entry event is linked to plasmid DNA entry.
There are now a number of methods of linking DNA to carrier adenovirus. A
versattle method mvolves btotinylatmg the adenovrrion and linking the plasmid DNA with a streptavtdin-polylysine bridge (6). Other methods include
direct chemical linkage between virus and polylysme (I&14), as well as an
enzymatic linkage using transglutaminase (15,16). The drawbacks with the
direct virus-polylysine linkage methods are the generation of a fixed virus-topolylysme ratio (limiting titration posstbilittes), and the storage difficulties of
polylysme-adenovuus that necessitate the use of high tonic strengths. A coupling technique using antibody-polylysine binding to an adenovirus epitope
has been described that allows a greater degree of flexibility m vnus-polylFrom
Methods
EdGxl
m Molecular
Medune,
Vol
21. Adenowrus
by W S M Wold 0 Humana
295
Methods
and
NJ
Protocols
296
ysme ratios (13, but requires both an epitope-tagged virus and a special antibody-polycation conjugate.
A number of reviews on these systemsare available. These mclude reviews
on receptor-mediated gene-delivery systems (18-20); on adenovirus-augmented systems (15,21-23), and reviews on general nonviral gene delivery
systems (24,25).
We descrtbe here a simple method of linkmg plasmid DNA to carrier adenovnus particles. The method uses the synthetic polycatton polyethylenimine
(PEI) to condense the plasmid DNA into a small, postttvely charged complex.
PEI m a high-molecular-weight form has been shown to possessDNA transfer
activity (26,27). We initially found that PEI was useful in preparing gene transfer complexes with high-molecular bacterial arttticial chromosomes (BACs)
when polylysme was found to generate precipitatton problems with these large
DNA molecules (28). Further mvestrgatron of PEI properties revealed that PEU
DNA complexes could be bound ionically to the negatively charged adenovirus type 5 capsid and these DNA/PEI/adenovnus complexes transfected with
efficiencies comparable to the original streptavidm-polylysine complexes (29).
This transfection system is illustrated in Fig. 1. The PEI system is inexpensive
and the ready availability of the reagents make tt simple to establish.
In addttion to descrtbing the PEI plasmid DNA/vn-us linkage method, this
chapter also describes the preparation of psoralen-macttvated carrier adenovirus. Furthermore because the plasmid-DNA purity with regard to LPS is crucial, especially when transfecting primary-cell types, a simple method for
removing LPS from plasmid DNA is provided.
2. Materials
2. I. Psoralen Inactivation of Adenovirus, Quantitation
1. 8-methoxy psoralen.(Sigma, St Lotus, MO, cat. no. M-3501) P A stock solutron
2
3.
4.
5.
6.
Transfection Complexes
297
Formation of PEIiDNA/adenovirus
polyethylenimine
r;\
complexes
adenovirus
negative
PEIw
fNH-CI$-CH&
DNA
PEIIDNA
net positive
charge
PEUDNA/adenovirus
Fig. 1. DNA is complexed with PEI to generate 300-nm, positively charged complex. Adenovirus is added to the DNA complex and negative charges on the hexon
bind positve charges on the PEI/DNA. This results in 400-600 nm transfection complexes. For additional details, see ref. 29.
298
stock solutton was stored at 4C Before use, the 10 mM PEI stock solution
was vortexed vigorously.
2. Transferrin-polylysme (from Boehringer Ingelhelm, Austria).
3 DMEM (no serum) (Gibco-BRL, Galthersburg,MD, cat no 31600)with 3 7 g/L
NaHCO,, 2 mM L-glutamme, 100IU pemclllm, 100 pg/mL
4 DMEM 10% FCS. (Glbco/BRL cat. no. 31600) with 3 7 g/L NaHC03, 2 mM
glutamme, 100 IU penicillin, 100 pg/mL streptomycin and 10% (v/v) fetal calf
serum.All seraareheatinactivatedat 56Cfor 60 mm before addltlon to medium.
3. Methods
3.1. Psoralen Inactivation
of Adenovirus,
Quantitation
3. I I 7. Choice of Adenovirus
When psoralen-inactivated virus 1sto be used, the genotype of the vn-us IS
not critical because all gene expression from the virus is extinguished by the
psoralen treatment. Two other considerations can influence the choice of virus
One practical consideration is the growth of the virus. Adenovn-us serotypes
such as Ad2 or Ad5 or CELO that can be easily and rapldly grown to high titer
wtll facilitate preparative work. Safety conslderatlons also suggest the use of a
genetically defective vtrus so that the combined genetic defect and the psoralen
inactivation give two blocks to virus replication. We normally use the E4defective Ad5 dl1014 from Gary Ketner (31) grown on the E4-complementmg
cell line W 162 (32) The virus grows nicely, and the complementing cell line IS
very easy to handle.
Psoralen mactivation of adenovirus was developed as a method of disrupting the vn-al genome without lmpalrmg the viral capsld and the virus entry
properties (33). Whereas other methods of vnus mactlvatlon exist (short wavelength UV light, heat inactivation, formaldehyde treatment), these methods can
severely damage the protein component of the virlon as much or more than the
nucleic acid and thus generate vlrions that are no longer competent to enter
cells (and are no longer useful for codehvery of DNA). Psoralen enters the
capsld and intercalates in the viral DNA. Upon exposure to long-wavelength
UV light (360 nm), covalent psoralen/DNA crosslinks are generated that effectively block DNA replication and RNA synthesis from the viral template
The methods described in this chapter employ CsCl-purified adenovirus
particles. Pure adenovu-us IS essential for several reasons, Because the vu-us 1s
eventually used m a psoralen-inactivated form, it is not possible to quantltate
the virus by plaque assayor other assaysthat measure the infection or replication properties of the virus. Therefore, physical methods to measure virus particle numbers are used and these requtre that the only protem or DNA m the
preparation 1sfrom the virus particle. A detailed protocol of the CsCl-punficatlon method used m our laboratory has been published (16).
Transfection Complexes
1. Place purified adenovirus in HBS/40% glycerol (300 l&/well) m a 4-well tissue
ceil-culture dish (NUNC cat. no 176740) or 24-well dish (NUNC cat no
143982). Other adenovirns-storage buffers, for example, containing sucrose, or
vuus directly from CsCl gradient should also be suitable
2. Add an aliquot (1.5 pL) of 33 mg/mL 8-methoxy psoralen (m DMSO) to each
300-pL sample and the psoralen and virus are incubated for 20 min at room temperature The room-temperature incubation facilitates penetration of the virion
by psoralen A psoralen precipitate forms immedtately. This is normal and does
not interfere with the mactivation This concentration is at the solubility bmtt of
psoralen Curiously, however, lower concentrations of psoralen are not as effective in inactivatmg adenovuus.
3. Place the plate on ice (wtth cover on), 3 cm below the filter of a 365nm light
source (UVP model TL-33) The plastic cover functions as a filter, blockmg
shorter wavelengths of UV light that could be absorbed by and damage the vmon
capstd protems
4 Irradiate the plate for 25 min, repositioning the samples every 10 mm to ensure
that no virus remains m a shadow.
5. Remove unmcorporated psoralen by passing the virus over either a Pharmacla
PD-10 gel filtration column (for 2 mL volumes) or a Nick column (for 400 pL
volumes) equilibrated with HBS/40h glycerol. Dialysis is not an effective
method of removmg free psoralen.
6 Quantitate the purified virus by protein or DNA measurement (see below) and
freeze the virus rn aliquots at -70C.
300
5. The vu-us parttcle concentration is determined
1 mg/mL protein = 3.4 x lOI2 virus particles/ml
Transfection Complexes
301
302
4. The sample is incubated at room temperature for 20 mm. Add an aliquot of adenovnus (1 5-5 pL, 2.5 x lo9 particles/pL in HBS [ 150 mMNaCl,20
WHEPES
pH 7.41 containing 40% glycerol) to the PEI/DNA
5. After an additional 20 min at room temperature, add ahquots of the complex to
cell culture medium without serum (e.g., 50 pL of transfection complex for
250 pL of medium covermg 20,000-50,000 cells m a 1.5-cm diameter well, e g.,
the well of a 24-well dish). This is the maximum dilution to be used for the PEI/
DNA/adenovnus complexes Unlike the streptavtdm/btotin
adenovnus complexes (6), the PEI/DNA/vnus complexes are sensitive to dilution.
6. Followmg a 4-h mcubatton at 37C replace the transfection medium by fresh
normal medium, at optimum volume and FCS concentration.
7. The harvest of cells and analysts of gene expression can begin as early as 12 h
posttransfection. Optimum gene expression depends on the cell type, promoter,
and gene, and should be determined emptrically.
4. Notes
4.1. Parameters
1. Titrate the level of PEI m the system, keeping the DNA constant. Small uncertainties m the PEI concentration and in the DNA concentration may exist and a
modest titration can generate useful improvements.
2 Vary the quantity of adenovirus used, keeping the PEI and DNA constant. Large
quantities of adenovirus (e.g , 10,000 particles/cell) give very good gene delivery
results at 24 h, they can result in a toxicity at later times More modest virus
levels can give useful gene delivery at later times One limitatron with the PEI
system relative to the streptavtdm/btotm or chemical linkage methods is that the
PEI/DNA complexes are very senstttve to dilution and the PEI/DNA mteractions
reverse rapidly upon dilution. Therefore if one wants to lower vnus/cell ratios It
1sbetter to keep the PEI/DNA constant and add less vtrus to the complex, rather
than to simply add less of a PEI/DNA virus complex.
3 If no gene expression is obtained and the treated cells appear similar to untreated
controls (density, proliferation), this suggests that the transfection complexes are
not entering the cell. There are several reasons why this might happen
a. The cells are too confluent because endocytosis and vnus entry are downregulated m many cell types upon confluence. Be certain that cells to be transfected are not too confluent at the time of transfectlon However, confluency
seldom results in a complete block to transfection success.
b The adenovirus preparation is poor. Adenovirus stored m the absence of glycerol or sucrose can lose entry functions upon multiple freeze/thaw cycles Use
of a short-wavelength UV lamp rather than the 350-nm lamp can damage the
protein and entry capacity of the virus.
c. The cell type is not susceptible to entry by the adenovirus serotype used.
d. The DNA concentration 1s incorrect. The wrong DNA:PEI ratio can result m
preciprtatron of the complex.
Transfection Complexes
303
4.2. Applications
of Method
5. The use of commercially available PEI allows investigators to prepare plasmid DNA/adenovnus complexes with hgh-transfwtion efficiency quickly and without exotic reagents.
6. Thehostrangeofthevn-us complex can be altered by including commerctally avallable transferrm-polylysine in the complex (add 0.5 pg of transferrin-polylysine
to the PEI solution before adding the PEI to the 6-pg DNA sample). This modlfication has been used to increase Ad5 transduction of hematopoletlc cells or to
Increase avian adenowrus
7.
8.
9
10.
11.
transduction
have been used with adenovirus polylysme DNA complexes. For example, antibodies to CD3 have been used to increase T-cell transduction (42), transferrm or
lectms have been used to enhance chlcken adenovlrus entry into mammalian cells
(43), and lectms or antIbodies that recognize carbohydrate motifs have been used
to increase tumor cell entry (44,45).
The reagents described here are very good for generatmg high levels of transient
gene expression m primary cells. This has been used for the rapid modification of
human tumor isolates with cytokme genes in efforts to generate a tumor vaccine
(46). Gene correction experiments using fibroblasts derived from gene-knockout
mice have been facrhtated by the high-transfection efficiency of these complexes
m primary mouse fibroblasts (47).
Coupled plasmld copies of adenovlral genes have been used to complement
defective adenovuuses and allow transient, local replication of the virus (48,49).
Transient overexpression of retrovlral packaging functions using these complexes
allow rapid production of retrovlral stocks (50)
This system has been used to identify a novel antiapoptotic gene in the avlan
adenovuus CELO (51)
The system can deliver large DNA molecules. We have had success delivering
170 kb BACs (bactenal artificial chromosomes, ref. 28).
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with HeLa cells: penetration of type 5 and mtracellular release of the DNA
genome. Vu-ology 40,462-477.
35. Mtttereder, N., March, K. L , and Trapnell, B C. (1996) Evaluatton of the concentration and bioactivity of adenovnus vectors for gene therapy J Vwol 70,
7498-7509.
36 Lemay, P., Boudin, M., Milleville,
M , and Boulanger, P (1980) Human adenovirus type 2 protein IIIa. I Purification and characterization Vzrology 101,
131-143.
37. Cotten, M., Baker, A, Salttk, M., Wagner, E., and Buschle, M. (1994)
Lipopolysacchartde
IS a frequent contaminant of plasmid DNA preparations
and can be toxic to primary cells m the presence of adenovirus. Gene Ther 1,
239-246.
38. Cotten, M. and Salttk, M. (1997) Intracellular
39
40.
41.
42
43.
44
45
46.
delivery of LPS during DNA transfectton activates a lipid A-dependent cell death response that can be prevented by
polymyxm B. Human Gene Ther 8,555-562.
Manthorpe, M , Cornefert-Jensen, F , Hartikka, J , Felgner, J , Rundell, A ,
Margahth, M., and Dwarki, V. (1993) Gene therapy by mtramuscular inJection of
plasmid DNA: studies on firefly luciferase gene expression m mice Human Gene
Ther 4,419-431
Aida, Y. and Pabst, M. J. (1990) Removal of endotoxin from protein solutions by
phase separation using Trtton X-l 14. J Zmmunol. Methods 132, 191-195
Cotten, M., Wagner, E , and Bnnstiel, M L (1993) Receptor-mediated transport
of DNA mto eukaryotic cells. Methods Enzymol 217, 618-644
Buschle, M , Cotten, M., Knlappos, H., Mechtler, K , Schaffner, G , Zauner, W ,
Birnstiel, M. L., and Wagner, E (1995) Receptor-mediated gene transfer mto human T-lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T
cell receptor complex. Human Gene Ther 6,753-76 1
Cotten, M., Wagner, E., Zatloukal, K., and Birnstiel, M. L. (1993) Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammahan
cells and yield exceptional levels of stable transformants. J Vzrol. 67, 3777-3785
Thurnher, M., Wagner, E , Clausen, H , Mechtler, K , Ruscom, S , Dmter, A ,
Berger, E., Birnsttel, M., and Cotten, M. (1994) Carbohydrate receptor-mediated
gene transfer to human T-leukemic cells. Glycobzology 4,429-435.
Batra, R. K , Wang-Johanning, F., Wagner, E., Garver, R. I , and Curiel, D T
(1994) Receptor-mediated gene delivery employmg lectm-bmdmg specificity
Gene Ther 1,255-260.
Zatloukal, K., Schneeberger, A., Berger, M., Schmidt, W., Koszik, F., Kuttl, R ,
Cotten, M., Wagner, E., Buschle, M., Maass, G., Payer, E , Stingl, G , and
Btrnstiel, M L. (1995) Elicitation of a systemic and protective antimelanoma tmmune response by an h-a-based vaccine. assessment of critical cellular and molecular parameters. J Immunol. 154, 3406-3419.
Transfection Complexes
307
47 Schrerber, M., Baumann, B., Cotten, M., Angel, P , and Wagner, E. F (1995)
c-Fos is an essential component of the mammalian UV response. EMBO J 14,
5338-5349.
48. Goldsmith, K T., Currel, D. T., Engler, J. A., and Garver, R. I. (1994) Trans
25
Phylogenetic
Analysis
of Adenovirus
Sequences
309
and Protocols
NJ
310
2.1. Hardware
The usual laboratory computers: IBM-compatible 386s or 486s (with or
without mathematical coprocessor), Macinthosh, or UNIX-based computers
will suffice. However, having more power (Pentmm processor) and memory
(e.g., 16 MB instead of 8) will yield speed. This may be important, e.g., m
bootstrap calculation (see Subheading 3.3.3.1.), when distance analysis with a
Phylogenetic Calculations
311
normal 100 data set sampling of a 900-residue-long ammo acid sequence may
take 10 h on an IBM 486DX2/66 MHz computer.
2.2, Software
We have used the PHYLIP (Phylogeny Inference Package Version 3.5~)
written and freely distributed by Joseph Felsenstein (22,22). It can be downloaded by anonymous ftp. The procedure to obtain the program package
through the Internet, and run It 1sdescribed in detail in Subheading 3.
We describe below the use of the version for Windows 3.1. Some small
differences in the use of the program package on platforms DOS and Win95
can be found m Subheading 4.; usage of the Macintosh, PowerMac, and UNIX
versions 1sdescribed m the dot files of those versions.
3. Method
You will find interesting information about this and similar phylogenetlc
programs, and a menu selection will take you to a page to fetch the necessary
files. I will assume that you want to run the program under Windows 3 1. It is
clearly shown that you have to click on the following files:
Documentation an; C source code
386 Windows executables, part 1
386 Windows executables, part 2
386 Wmdows executables, part 3
Save these files on your computer m a location you will have accessto later.
3.1.2.
312
Windows program files are. If your computer is a 386 compatible without math
coprocessor, you also have to deal with an emulator program of the package
according to the description found in the readme.wm text file Rename font2 (or
your choice of font from the offered six types) to be called font f i 1 e
5 Start the wmdows (win), and open the group tile (click on Frle, New, Group of
files, give the name Phylip
3 .5c,
<O.K.>). You will see 30 icons (programs) in a window called Phylip 3 5c
significant data, the length of the sequences should be over a certam limit. The
required
minimal
to the heteronenertv
of the studted
Srmilarly,
pVI1 seems to be
useless for this purpose because its amino acid sequences are not only quite
short, but they are also constderably heterologous, and then homologous parts
(applicable in the analysis) are too short.
For an easy start, a comparison of amino acid sequencesmay be suggested.
It is easier to obtain and align them, and they seem to give more explicit patterns
than DNA sequences.To find all the available homologous sequences(or to check
if there are new ones submitted that you are not aware of), you may send a query
to GenESank.
A simple e-mail will let you know all the available sequences(or ensure
that you have got all of them). You can send the chosen protein sequence,e.g., to
the heurtstic homology search program blast (23,24) of the National
Biotechnology
Information (NCBI), m the form as follows.
PROGRAM blastp
DATALIB nr
BEGIN
>any name to identify
your sequence
MGSSEQELKAIVKDLGCGPYFLGTYDKRFPGFVSPHKL......
Center for
Phylogenetlc Calculations
313
on the previous page.Gtvmg a name to the received file wtth an . htm extension and lookmg at it by a www browser program (e.g., Netscape), the accession numbers will be blue. You simply click on any of them, and Netscape
gets the sequences rtght away. To get any sequence by simple
sendane-mailcommandto
retrieve@ncbi.nlm.nih.gov:
DATALIB gb
BEGIN
<accession number(s)>
e-mall,
Just
(or sp /=SWISS-PROT/)
314
results to be displayed. This means that there are different routes to perform
the complete analysis, and some of them are depicted u-rFig. 1.
3.3.1. Alignment
Since only homologous regions can be studied, the sequences must be
aligned. For this purpose, many multtple alignment programs are available.
Clustal V (25,26) or its new version Clustal W (27) are freely distributed. The
Internet may be used to get them (http : //www . ebi . ac . uk/). However,
most probably, there are some general sequence-analysts programs available
m your mstitute, e.g., on-line from a university server In the Wisconsin program package GCG, you may use the PILEUP. The Lasergene package of
DNASTAR offers MEGALIGN. PC/Gene has Clustal V (Fig. 2A) Of course,
there are differences m the calculatton and the accuracy of the different alignment programs; e.g., during the multiple alignment, terminal gaps are penalized in Clustal V but not m PILEUP This ~111make the PILEUP alignments
better when the sequences are of very different length (has no effect if there are
no large terminal gaps).
It is advisable to start the alignment with the HAVs (our favorite is the completely sequenced HAV-2), as there are so many sequences from the HAVs
that it is easy for the program to make consequent alignments. In the lme of the
sequences to be aligned, the most exotic ones should be at the end, so at the
time of their alignment to the other sequences,the program would have already
a quite good consensus to align to. The alignment has to be saved into an
editable file that will be used to create a specially formatted tile for the phylogenetic programs. As a preferred optton, this file should be named inf ile.
If DNA sequencesthat code for proteins are aligned, one should first make a
protein alignment to ensure that triplets coding for identical ammo acids are
aligned in the DNA alignment, as well. Practically, this means that the size of
the gaps m a DNA sequence alignment can only be a number dividable by
three. In the case of less stmilar proteins (where more gaps occur), one may
experience many mistakes in the alignment. Manual correction and constant
comparison to the protein alignment make this work much more time consuming than to analyze protein alignments. Making DNA alignment is also more
complicated because the DNA sequences contained in databanks are usually
longer then the actual genes.
Fig. I. (oppositepage) Flowchart of different typesof phylogenetic analysisby the
use of PHYLIP programs. Continuous line shows single alignment analysis, dotted
line depictsanalysiscombined with bootstrapping.The useof programs typed in bold
IS highly recommended, the use of other programs is optional. The necessary
renamingsare shownas DOScommandswritten in Courier. Options to be changed
Phylogenefic Calculations
315
CLUSTAL
1. Alignment
2. Edlting
tormat
rename hexon.txt
3. Analysis:
infile
Bootstrapping
Parsimony
analysis
Distance
matrlx
..
3
**
*.
\
DNADIST
PROTD:ST,
analysis
6
&<iple
data)
rename outf?le
t
FITCH
&lobal
realrangements)
&jultlpb : data)
Consensus
Drawing
infile
i
I
calculation
the tree
Rearranglng
the tree
REkiEE
\\
(0, [Jl 0, /k Tl Xl U)
4
rename outtree
treefi
4. Editing
figure
Insert
values
as a picture,
of CONSENSE
type
I
ie
the bootstrap
etc.
Fig 2.
Phylogenetic Calculations
317
Fig 2. Multiple alignment of the amino acid sequences of known adenovnus proteases (A) Given by the program Clustal V (B) Edited mto PHYLIP format. Aligned
sequences and their accession number in GenBank or EMBO database: BAV- 1 (B 1)
(manuscript in preparation, sequenced by Peter Evans); B2, U44124, B3, X53990; B4
(ref. 16; manuscript m preparation); BAV-7 (B7), X53989; canine adenovirus type 1
(Cl), M72715, EDS virus, U63515; fowl adenovirus type 1 (Fl), L13161, HAV-2
(H2), 501917; H3, X13271, H4, M16692; H5, M73260; H12, X73487; H40, L19443;
H41, M21163; murine adenovirus type 1 (Ml), M33995, ovine adenovirus isolate 287
(OAV287), U40837; ovine adenovirus type 3 (03), (manuscript m preparation,
sequenced by Cyril Barbezange), porcine adenovirus type 3 (P3), U33016.
318
3.3.2.1
This is the artistic step of the analysis, you have to use your Judgment, and
no exact rules can be given on what to include into an analysis. However, do
not worry. If the sequences are long enough, and the differences you want to
demonstrate are significant, then even quite big changes m the selection of the
sequence will result in similar evolutionary trees. Actually, this IS the basis of
the bootstrappmg (see Subheading 3.3.3.1.), when you are sampling different
parts of the alignment to test if they are yielding the same tree or not.
Our usual policy 1sthe removal of any region suspected to be nonhomologous based on the observations that:
1 Deletions (gaps) of more than four to seven residues are present The reason for
domg this 1s that a deletion may be the result of a single event, and It should not
be counted as an independent mutation for every smgle residue On the other
hand, some (smaller) deletions maybe very conclusive, showmg deletions
appearing identical m closely related vnuses, e.g , the same three-residue-long
deletion m BAV-4, BAV-7, OAV287, and EDS viruses (candidate members of
the proposed new genus) (Fig. 2B).
2 Ahgnment regions do not have homologous residues for a length of more than
five to eight residues (i.e , you do not see any resemblance, not a single amino
acid that 1s the same m at least some of the sequences)
3. Some sequences are longer than the others. The analysis should be ended at the
stop codon of the shortest sequence. The stop codon may be represented by an
asterisk. (Theoretically, there IS an optlon to put questlon marks after the last
residue lmplymg that anything could be there if it were not ended, but m our
practice this resulted m mlscalculatlons ) A different case 1swhen obtammg preliminary results, where a short sequence IS compared to full-length sequences. In
this case, some question marks may be Included (not gaps, because they mean
existing deletions), yet the sequences must be abbreviated almost to the size
of the shortest sequence. Analyzing both the short alignment (with the added
new sequence) and the long alignment will show the sigmficance of the results
gamed with the short alignment. The detection of large phylogenetlc differences,
e g., genus differentiation 1s usually no problem even with short sequences
319
residues 118-122. Smaller deletions were made at the start and at the end
of the alignment.
On the other hand, the hexon or the DNA polymerase protems are very long,
and they have some highly variable regions. Aligned amino acid sequences of
the hexon and polymerase (and protease) can be found m our personal
databank (mainly on Adenovzridae) named GeneFarm and available through
www(ht tp ://www.vmri.hu/-(harrach).
Youmaydownload,edit,and
test these alignments on your own computer. This way you may test the effect
of different deletions, or if you have sequence from another serotype, you may
compare and analyze it Because of the variable regions, both the polymerase
and the hexon sequences had to be deleted extensively to consider the resulting
sequences homologous. Our final alignments of hexon and polymerase ammo
acid sequences can also be found on the GeneFarm pages m ready to analyze
PHYLIP mfile format.
3.3.2.2.
PHYLIP
FORMAT
The data prepared for analysis have to start by the number of aligned
sequences/speciesand the number giving the total length of the sequence alignment (these numbers are 19 and 197 in Fig. 2B). The molecular sequence programs (described m our examples) can take the data in mterleaved (aligned)
format. Each sequence starts m a new line, has a IO-character species name (if
shorter it must be blank-filled up to ten), followed immediately by the ammo
acid sequence m one-letter code (Fig. 2B). The blocks of sequences can be
separated by an empty line. This format is similar to that yielded by most of the
multiple alignment programs, but the names can be present only in the first
block, and no sequence numbering and signs showing the homologous residues are allowed. The blocks do not have to have the same length, so the
nonhomologous regions can be deleted. Blanks are allowed, e.g , at every tenth
position. The blank lme separating the blocks should not contam any extra
blank character. Non-IUPAC abbreviation (e.g., end of tile symbol: //) in the
sequences or blanks at the ends of the lines are not allowed.
In an alternative format called sequential, data are entered continuously one after the other. This format is obligatory for certain PHYLIP
programs (gene frequencies, discrete and quantitative characters programs)
not described herein
A simple editor (e.g., Norton Commander) might be efficient when working
with short sequences or introducing minor changes. For longer sequences or
extensive editing, however, more sophisticated word processors should be
used. It is easier to remove every name after the first block, and all the numbers
after the sequences by deleting columns (Control+Schift+F8 m Word for
Wmdows). When using Windows based word processors, single spacing
320
and proportional font (i.e., characters of uniform width, e.g., Courier New)
should be applied. The results should be saved as Text Only with Lme Brakes
(ASCII format).
3.3.3. Performing the Analysis
During Windows 3.1-based program operation, the user 1s required to go
back to DOS sometimes. The tree drawing programs run under DOS, but it 1s
not noticeable once the program has been successfully loaded. However, DOS
1squicker m the necessary renaming and checking of simple text files, and in
printing out the results. Ltfe can be made easier by placing MS-DOS or preferably a Norton Commander icon right mto the PHYLIP window. Cltckmg on it
allows to find, rename, copy, or look at the files. Typing exi t (or FlO If Norton
Commander icon was used) returns the user to Wmdows. This constant platform changing between DOS and Windows can go routinely. Alternatively the
Windows facllltles can be used.
The necessary steps to perform different calculations and to print out the
results are depicted m Fig. 1. To start quickly, the easiest way is to rename
your edlted file to infile, because the program will automatically look for such
a file. (However, any other file name can be specified.) After starting Windows, the programs can be performed by clicking on their icons one by one.
Between two steps, one always has to create the new mfile for the next program, I.e., usually the outfile 1srenamed for being the next mfile. In some
steps, It is advisable also to save the outfile or the treetile (the file which
contains the data to draw the tree). Loss of files (by overwntmg) can be prevented by copying them mto separate subdn-ectorles (and renaming it), because
each program 1screating outfile or/and treefile and plotfile, so the earlier results
will be overwritten.
3.3.3.1.
BOOTSTRAPPING (OPTIONAL)
Bootstrapping is used to give some statistical validity to the data after the
first round of phylogenetic calculations (Fig. 1). This program wrll use certain
columns of the alignment (even several times) while deleting others. Generally, 100 such random samplings of the alignment are analyzed to study what
changes in the results would occur if only certain (different) parts of the alignment were analyzed Ultimately, the CONSENSE program will count how
many of the 100 samplings yielded the final result.
Click on the icon of SEQBOOT. The program will prompt you to choose an
odd number (more exactly: 4n+l), and type m the requested number of
samplings (usually: 10 0).
Rename the resulted outfile to infile. Such renaming will be described from
now on in the form ofDOS orders, i.e., rename outf ile inf ile.
321
Phylogenetic Calcfflations
3.3.3.2.
PHYLOGENETIC
CALCULATIONS
outfile
infile
Tree building (FITCH): Click on FITCH (for the Fitch-Margohash calculation) or alternatively try KITSCH (less consequent results, but a demonstrative
molecular clock approach) or Neighbor-joining (less reliable). Select Multiple analysis if bootstrappmg is applied (M). We strongly suggest to apply the
Global search option (G). It results m removmg and re-adding each possible
group after the last species has been added to the three. This method increases
322
the reliability of the results, because the position of every species 1sreconsidered. The only disadvantage could be that It triples the run time. In case of
multiple analysis, calculate the consensus(below), otherwise proceed to drawing the tree.
3.3 3.3. CONSENSUS CALCULATION
If bootstrapping has been applied, this program calculates the most probable
tree (based on all samplings) and gives the bootstrap values. The tree of the
output file (outfile) has at each fork a number indlcatmg how many times
the group conslstmg of the species to the right of (descended from) the fork
occurred. If the sequence IS long enough and the evolutionary distance 1s
expressed, then the same clustering of the vn-uses would occur 95-100 times
from the 100 sampling These data can ensure that the selected sequence was
adequate for this type of analysis. When the bootstrap value 1s much lower
(below 70), then the tree topology 1suncertain. In such case, the genetic dlstance IS too small for definite conclusions. The analysis may be repeated with
longer sequences.
rename
treefile
infile
Click on CONSENSE
After a calculation, you may be surprised (upset) by finding the orlgmally
requested outgroup species (HAV-2 m our example) being not the first one (at
far right) in the parsimony tree but the last, To prevent the change of species,
change manually the Qutgroup: type 0 and the total number of the species
(because that will be the number of the species originally first). (For explanation, see Subheading 4.5.2.). If you want any other species to be the outgroup,
find out its number from theflrst tree of the treefile (from left to right). You
may alternate the appearance of the tree later, too, with the program RETREE
(see Subheading 3.3.5.).
3.3.4.
These programs use the created treefile, and write the created picture mto a
so-called plotfile.
3.3.4.1.
The gram format of PHYLIP is the classical tree format (Fig. 3A), which
seems to be a so-called rooted tree (with known direction of the evolution).
However, our calculations yielded unrooted trees. Yet, one can (has to) choose
an outgroup (i.e., a sequence where the tree is started). This should not cause
any problem, as long as we remember that in reality the tree is unrooted (this
fact should also be noted m figure legends for example). As we are mainly
Phylogenetic Calculations
323
1 Aviadenovirus
1
Mastadenovirus
cc2
Y----L
ATadenovirus
proposed
genus/name
324
interested in the characterization of animal adenovlruses, we hke to use HAV2 as outgroup. This way, we can see how the animal adenovnuses are related to
each other. However, one interested in the genetic distances between HAVs
may get a more easily interpretable tree by using a very distant virus, like fowl
adenovlrus type 1 (FAV-1) for outgroup.
The gram format tree (e.g., a phenogram) shows which speciesare clustered
together. The advantage of the gram format (vs tree format, Fig. 3B) might be
that the names appear at exact distances from each other, so they can be easily
read (whereas in the tree format they might be superimposed). Since the
PHYLIP parsimony programs cannot generate different branch lengths, we like
the gram format to view the parsimony results.
Click on the DRAWGRAM icon. A selection of output formats will be
offered. If you have a printer capable of handling Postscript format, choose the
L (Laser printer) to gain the picture (the plotfile) in Encapsulated Postscript
format. You may prmt out the result right away m a perfect format (print
plot f i 1 e). However, if your printer does not handle Postsript format, even
viewing on the screen is not possible. Select another available printer type (e.g.,
Epson), or the .pcx format (P). The .pcx (the PC Paintbrush format of
Windows) IS the only format of the PHYLIP program package that 1s
editable. It can be important if one wants to add anything (e.g., bootstrap values) to the picture.
There are many types of the gram format m DRAWGRAM. Our favorite is
the Phenogram, which means only a certain type of tree drawing, and not a
calculation type (2 P). We also prefer to select the weighted format (10 W).
Test the different formats to choose one for your taste.
3.3.4.2. DRAWING A TREE (DRAWTREE)
The tree format is an unrooted tree, like a real tree seen from above (Fig.
3B). It shows only the found similarities and differences, and IS not suggesting
any (possibly false) direction of evolution. This is our favorite representation
format (a must for the distance analysis results), as It can represent the genetic
distance by variable branch length. Certain theories can be clearly demonstrated, such as the distance between the clusters of the proposed ATadenovnus
group and the classical mastadenoviruses 1s as large as the distance between
the clusters of the two officially accepted genera (Avzadenovirus and the
Mastadenovirus).
The ATadenovn-us cluster therefore could be considered
equivalent to a genus.
3.3.5. Changing the Appearance
For cosmetic reasons, the resultant tree can be rearranged, the order of the
species on a branch can be inverted; e.g., subtrees can be flipped (rotated) at a
Phylogenetlc Calculations
325
node, the outgroup species can be changed (rerooted) m an unrooted tree, and
unnecessary species can be removed (e.g., when temporarily confidential data
were used). None of these changes is cheating, because a certain tree topology
can be shown different ways. The length of the branches and their order must
stay fixed, but flipping of certam branches does not influence the result. Any
mistakes in the names can be corrected (no need for complete recalculations).
rename treefile
intree
Click on RETREE. Accepting the default values, you may get an unreadable
junk. Change the terminal type by typing 0 (zero). Accept the options now (Y),
and the tree appears. If the tree 1s larger than the screen (N lines below
screen), type J to see the end of it. You may have to repeat the typing of J. (If
you want to see agam the top of the three, type K.) Note the number of the node
you want to flip or to choose as outgroup. Type 0 (letter 0) to reroot, and the
present node number of the species (or whole branch) requested to be the new
outgroup. (Finally, the newly chosen outgroup will be on the left side of the
phenograms, not as drawn after the earlier programs.) Typmg F or T, and a
node number can flip/rotate the subtree or transpose the immediate branches at
a chosen node, respectively. Quit the program by typing X (esit) and U (tree
remains unrooted)
3.3.6. Editing the Picture
If the picture has been saved m .pcx format, it can be inserted mto a Word
for Windows (Winword) document as picture, and by clicking on the drawing
tcon of Wmword 6.0/7.0, it can be further edited. In the case of distance analysis, one may want to write the bootstrap values into the figure of the earlier
analysis of the complete alignment (but not on the tree produced by
CONSENSE), because CONSENSE cannot give branch lengths. The bootstrap
values can be found m the saved outfile of CONSENSE, and typed to the right
forks. The most important value can be colored/enlarged/boxed as is number
100 on Fig. 3 showing that the ATadenovlruses were always clustered together
(after both parsimony and distance matrix analysis). The resulted (color) plcture can be printed, sent by e-mail, or displayed on your www pages.
3.4. lnferprefation of the Results
The unrooted trees show only the presently existing relation among the different adenovirus types. The directzon of the evolution cannot be seen. One
may, however, try to identify which of the presently existing serotypes might
have had common
ancestry.
The evaluation of the results may be started by analyzing which adenoviruses clustered together based on distance matrix analysis (pure slmllanties)
326
and also on parsimony analysis (identifymg which tree can be built usmg the
lowest number of residue changes). These types probably evolved together.
We have found that certain viruses clustered together independently of their
host species (BAV4, -7,0287, EDS vnus), whereas others clustered into two
addittonal groups according to their (mammalian or avtan) host. The three clusters were placed at a considerable distance from each other (Figs. 3-5) It can
be concluded that the phylogenetlc analysis detected the genetic distance between the Avladenovirus and Mastadenovirus genera with high confidence (as
expected). The data also suggested that there are altogether three zndependent
lineages
of adenovwuses
infectmg
the members
mammals, and the members of the thud lineage may infect both avian and
mammalian species.We propose to establish a new genus for the viruses of the
thud lineage. The third cluster comprtsmg the nontypical members of the
Mastadenovwus and Aviadenovlrus genera was placed as distant from any of
the two genera as they were from each other. We believe that the identified
evolutionary distance alone merits the establishment of a new genus. Unique
genome orgamzation and other characteristics of the members of this cluster
also support the distinction on genus level.
It is not the direct subject of this work to suggest an appropriate name for
this proposed new genus. Nevertheless, because this cluster cannot be related
to a single animal class (like the Mast- and Avzadenovzrus genera are), we propose to call it ATadenovzrus, a name descrrbing a sulking common property
(the very high AT content of the genome) of its members.
The following additional observations could be made. First, as expected, all
HAVs are very similar to one another, mcludmg HAV-40 and 4 1, in spite of
then distmguished characteristics (such as two fibers, restricted growth) compared to the differences found between several bovine adenovnus types. Obviously, all the HAVs isolated so far have a single ancestor and evolved
separately from the animal adenovnuses that have been studied.
OAV-3 was found to be closely related to BAV-2 (and to a lesser extent to
BAV- 1). The similarity between BAV-2 and OAV-3 suggests a recent adaptation to the other host (manuscript in preparation). The likelihood of their recent
separation IS supported by the fact that BAV-2 is capable of mfectmg ovme
hosts as well (28-30).
From the mastadenovnuses, the murme adenovmus always proved to be the
most distant relative of the HAVs.
The followmg animal viruses were confirmed as mastadenoviruses (I.e.,
bemg close relatives of the HAVs): BAV-I , -2, -3, CAV- I, MAV- 1, OAV-3,
PAV-3, and tupaia adenovirus 1.
We have to accept the fact that very close relationships cannot be resolved at
satisfactory confidence, and in this case, different genes or different programs
Phylogenetic Calculations
327
Fig. 4. Parsimony (A) and distance matrix analysis (B) of hexon sequences demonstrate the genetlc distance among the suggested three genera. Length of the sequences
taken as homologous parts and analyzed was 899 residues (the alignment and the
PHYLIP mfile are available through www). Aligned sequences: BAV-3 (B3), K01264;
FAV-1 (FI), 267970; FlO, U26221; HAV-2 (H2), J01917, H3, X76549, H5, M73260,
H7, X7655 1~H12, X73487; H16, X74662; H40, L19443; H41, X5 1783; H48, U20821,
MAV-1 (Ml), U81889; OAV287, U40837, PAV-3 (P3), U34592.
may yield contradictory results (see also Subheading 4.). The exact hmlts of
this method must be Identified specifically to the given questions. While all
methods have hmltatlons, consistent data should be accepted. Bootstrapping
328
Harrach
and Benkd
Mastadenovirus
was invented precisely to give statistical validity to the data. High bootstrap
values suggest reliable results, while low values (around 50) show the madequacy of the analysis to solve that special evolutronary relation (longer or/and
other sequences should be used). The constantly growing number of sequences
available for analysis will result in increasing rehabrlity.
4. Notes
4.1. Alternative Ways to Acquire
4 7.7. Direct ftp
the Programs
Use direct ftp when no direct Internet connection is available but there is,
e.g., an X.25 connection; or you experience special problems with www.
You start an ftp session from a computer that understands the f tp command:
ftP
open evolution.genetics.washington.edu
LOGIN:
anonymous
PASSWORD: <your-E-mail-address>
cd /wb/phylip/
Phylogenetic Calculations
329
binary
get phylip.exe
get phylipwx.exe
get phylipwy.exe
get phylipwz.exe
quit
4.1.2. Disks
The programs may be obtained also on disks from Joe Felsenstem or other
colleagues. The four self-extracting files necessary to run the programs under
Windows require four disks of HD/1.44 MB.
4.2. DOS and Win95 Versions
4.2.1. DOS
For PC/XT or PC/AT computers, get the three pre386 DOS self-extracting
files: phylip exe (C sources and dots), phylippx, and phyllppy (DOS
executables) For a 386 running only DOS, get phyllp.exe, and the 386 PCDOS
executables: phyllp3x, and phyllp3y. Copy them mto a (large enough)
subdirectory and start (unpack) all the three of them. The programs can be
started by typing their names.
4 2.2 Win95
The program can run m background in Win95 while doing other work m
other windows. The same files used by Windows 3.1 can be used by Win95.
All files, including the dos4gw and phylip.grp files should stay m C:\PHYLIP\.
Any of the programs can be run by clicking on the Start, then Run buttons,
typing C:\PHYLIP\<any-PHYLIP-program>
(e.g., Fitch),
and
<ENTER>. Alternatively, type C : \PHYLIP\, click on Browse, double click
on the icon of the requested programs and <OK> . Clicking on the phylip.grp
file will establish a new PHYLIP 3.5 menu item in your Start/Programs menu.
A permanent folder can be made for selected (frequently used) programs.
With the right button of the mouse click on the middle of the screen, then on
New and Folder, type Phylip
3 .5c, double click on the new folder icon,
chck somewhere in the resulted wmdow with the right button of the mouse,
click on New, then Icon, and Browse, double click on PHYLIP, double click
on the icon of the requested program (repeating this whole procedure for each
program), then on Next, and Fmlsh. Alternatively, open the windows of the
PHYLIP subdu-ectory and drag the necessary program icons to the new folder.
From this point on, the folder can be opened and the programs can be started by
clicking on them. A separate icon of Norton Commander can help to find, read,
and rename the files easily.
330
4.3. Organizing
Programs
and Files
It is advisable to copy all the .doc files mto a separate subdtrectory (e.g.,
DOC) m order to find the descriptrons more eastly, and also to decrease the
number of files m the PHYLIP dtrectory. It is very useful when one IS lookmg
for certain tiles. Any file ending by .c, .h, qc, .tc, pas extensions can be deleted
(as having executable files, there is no need to get them from the C sources).
The four origmal tiles can be moved onto 3.5 dtsks or onto a server, to gam
more space. Simrlarly, it ISvery useful to open another subdirectory m PHYLIP
for the mfiles and the resulted treefiles and plotfiles, the number of whrch ~111
rapidly grow. It is advantageous to give descriptive names for the gained tiles,
mcluding first the name of the characterized gene, and the type of analysis
(e g , for an alignment of &on sequencesanalyzed by &stance matrix analysis, bootstrapping included, the consensus output could be hexdl co. out).
4.4. Alternative Programs
4.4.1 Phylogenetic Programs
Several alternative programs are available. A very good collectron, descripttons, and drrect Internet connectrons to many of them can be found on the
PHYLIPwwwpages(http://evolution.genetics.washington.
edu / phyl ip . h tml). The PAUP 3.0 program package, written for
Macintosh (not distributed anymore, D. Swofford, Champaign, IL), was probably the most sophrstrcated parsimony program. Its new version will be available for both Macmtosh and DOS/Wmdows systems($100). However, our first
choice in demonstration of phylogenetic relations, the distance analysis (with
consistent results and branch lengths proporttonal to genetic distance) can be
performed perfectly with PHYLIP. PHYLIP also has the advantage of being
absolutely free, and can be obtamed within minutes, without the nuisance of
money transfer. It also contains a very large collection of alternative analysis possibihties (not described herem). It is the most widely distrrbuted
phylogeny package, with over 5000 registered users. By using these programs, you can better understand the lrmrtatrons or probabrhty of the results of
other scientists applying this package. (Please register, which IS a polite way to
express your gratitude.)
On the other hand, the hypothettcal phylogenetic tree (dendrogram)
obtained from the pan-wise similarity scores of Clustal V should not be considered as alternative for the results of the sophlstrcated phylogenetrc programs.
Clustal V forms the final multiple ahgnment only after makmg the dendrogram (beginning with the alignment of the sequences found to be most simrlar). This 1s the reason that the topology of the Clustal V trees may change
depending on the order of entermg the studied species
331
Phylogenetrc Calculations
4.4.2. Alternative Program to View, Rearrange, and Edit the Tree
copy phylip.grp
c:\win[orwindows]
and run It
b cant readinfile, give
sequence name>
c ERROR SEQUENCES
OUT OF ALIGNMENT
renamexwhatever-name>
infile
ortype
332
ERROR: END-OF-LINE OR
END-OF-FILE IN THE MIDDLE
OF A SPECIES NAME
J
Instead of renaming the outtile to
, infile, I gave outfile as sequence
name, but the program froze
1
ERROR: INCONSISTENT
NUMBER OF SPECIES IN
DATA SET 2
I ERROR CANNOT FIND
SPECIES. <second species>
Generally most of the errors are caused by a mtstake in the infile; check and
count it carefully.
4.5.2. I Wanted H2 to Be the Outgroup, but (After Bootstrapping)
It Is Elsewhere
After CONSENSE you will find that the originally requested outgroup specres IS the last; I.e., it is at the far left (at the top) of the phenogram instead of
being the outgroup at the far right. The reason IS that, after bootstrappmg, trees
are reanalyzed, and the CONSENSE program takes as outgroup again thefirst
species (default value). This first species IS actually the last one, i.e., the one
on the right of the tree format. As an example, see below the begmnmg of a
treefile, one out of 100 resulted trees (H2 was the first species and the selected
outgroup; it was always the last):
((((((((EDS,(B4,(0287,B7))),Fl),Ml),Cl),((B2,Bl),
W),Wl
,H40),((H4,H3),H12))),H5),H2),
To solve the problem, change the outgroup (0), select the last species, i.e.,
the one numbered by the total number of the studied species.
Phylogenetic Calculations
333
Each Other
03 and B2 are partly overlapping each other on Fig. 3B. Is there a solution
for this type of readability problem? One possibility is to turn the picture or the
direction of the letter by using DRAWTREE again with the original treefile.
To prevent such problems, long names should be avoided. DRAWTREE figures (including the sequence names) cannot be modified. (You could delete
them in certain picture editor programs, but it is not easy to write the same
type/size of letters to a better place.) However, if TreeView is applied, any part
of the Metafile figure can be moved or edited.
4.5.4. I Forgot Which Program Was Used to Create a Certain Picture
1. The analyzed viruses themselves will suggest a gene, e.g., (presently) if both
BAV-7
(3Z),
(Prmg-Akerblom,
2 The form of the tree can help to identify the applied program:
Drfferent branch lengths (either gram or tree format)-rt
was dtstance
4.6. Considerations
4.6.1. Selecting the Right Programs
Parsimony analysis calculates how many steps are needed to change a
sequence into the second one, then into the third one, and so on, and suggests
that the tree with the shortest overall length, i.e., with the smallest number of
required changes, represents most probably the past events. (Evolution, however, may proceed through more complicated ways.) Convergence (back
change) also exists, but it is not constdered by this sort of calculatton.
In the parsimony analysis of Joe Felsenstein, a change from one amino acid
mto another by the change of two nucleotides (both of them resulting m
codmg new amino acids) is calculated as two changes. (However, if one of
the two necessary changes was only a synonymous substitution, not ytelding a new amino acid, it is not counted because such synonymous changes
occur relatively frequently.)
Distance matrix analysis should be applied m parallel. It conststs of two
steps. First, every sequence is compared to all others producing a distance
Phylogenetic Calculations
335
sequences from BAV-1, -2, -4, -6, -9, -10, OAV-3, and EDS virus, though
many of these sequences are still preliminary and/or partial. Some practical
problems are listed m Subheadings 4.6.2.1. and 4.6.2.2.
4.6.2.1 NUMBER OF SPECIES
Some genes are more popular and well represented-eg., the protease, the
hexon, the pVII1 genes, and the inverted terminal repeat (ITR) regionswhereas others are available only from the four totally sequenced HAVs (HAV2, -5, -12, -40) and from FAV-1 and OAV287 at the submtssion of the
manuscript. The exclusive analysis of the completely sequenced viruses cannot give valuable phylogenetic results, since all HAVs cluster together, whereas
FAV- 1 and OAV287 are different (as logically expected because of the different host origin). Nowadays, the most comprehensive results can be seen from
the analysis of the protease, because sequencesare available from adenoviruses
of as many as eight species (cattle, chicken, duck, dog, man, mice, sheep, pig),
and the protein itself 1svery well characterized (37-39). It has also the highest
number of complete sequences* 19 presently.
4 6.2.2. INTERESTING" SPECIES
The different species are represented very unevenly; so if you are interested
m spectal questlons, you may have to choose different genes for the analysis.
1. Our original interestwas m BAVs, smce they were described to be very different
from one another. To be able to prove or to disprove the suggested difference
among them and the similarity among members of the two subgroups, we haved
sequenced many different types By now, we have the protease gene from five
BAV types (manuscript m preparation). This 1s the second highest number m
terms of representation of one host species (after the HAVs, of course) (Fig. 2)
Our results clearly proved the existence of very high genetic distance between
subgroups 1 and 2 of BAVs, and the existence of homology within the subgroups.
We plan to study further BAV types, especially the newest one (BAV-lo),
because of its questlonable subgrouping (4&42).
2. Some host species are represented only by rare sequences not available from
many viruses, therefore direct comparison cannot be done. Many slmlan adenovlrus sequences are avallable (32), but (with the exception of a partial SAV-30
polymerase sequence) (43) they do not contam useful late genes, but almost exclusively Just the VA RNA sequence. This was of little use in our comparisons,
since VA RNA has not been identified in ATadenoviruses (and 1s relocated in
aviadenovuuses). This demonstrates a special problem, that certain genes cannot
be used for the comparison of the members of all genera, if they do not exist m all
of them. In the case of underrepresented host species, there IS a pressing need for
further sequencing For example, the release of the hexon sequence of equine
adenovnus types 1 and 2 will enrich the picture (44).
336
Acknowledgments
We are obliged to Joseph Felsenstein and Rod Page for providmg then programs, to Krisztina Ursu and Adam Dan (Budapest) for makmg a comprehensive
collection of alignments and calculations of all available adenovnus genes, to
Peter Evans (Wisconsin), Miklos Rusvai (Budapest) and Cyril Barbezange
(Nantes) for providing amino acid sequencesbefore publications, and to Andrea
Ban&i and Gyorgy Berencsi (Budapest) for their help m sequencmg. Part of
this work was supported by National Research Fund of Hungary grant OTKA
TO16882 and TO21060, and T022405. Work performed at the Vetermary Medical Research Institute, Hungarian Academy of Sciences,Budapest, Hungary.
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