JOURNAL OF VIROLOGY, Jan. 1994, p. 346-351 Vol. 68, No.

1
0022-538X/94/$04.00+0
Copyright © 1994, American Society for Microbiology

A Single Chicken Anemia Virus Protein Induces Apoptosis

M. H. M. NOTEBORN,'* D. TODD,2 C. A. J. VERSCHUEREN,' H. W. F. M. DE GAUW,' W. L. CURRAN,2
S. VELDKAMP,3 A. J. DOUGLAS,2 M. S. McNULTY,3 A. J. VAN DER EB,' AND G. KOCH3
Laboratory for Molecular Carcinogenesis, Sylvius Laboratory, Leiden University, 2300 RA Leiden,' and Virology
Department, DLO-Central Veterinary Institute, 8200 AJ Lelystad, The Netherlands, and Veterinary Sciences Division,
Stormont, Belfast BT4 3SD, Northern Ireland2
Received 19 July 1993/Accepted 19 October 1993

Chicken anemia virus (CAV) causes cytopathogenic effects in chicken thymocytes and cultured transformed
mononuclear cells via apoptosis. Early after infection of chicken mononuclear cells, the CAV-encoded protein

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VP3 exhibits a finely granular distribution within the nucleus. At a later stage after infection, VP3 forms
aggregates. At this point, the cell becomes apoptotic and the cellular DNA is fragmented and condensed. By
immunogold electron microscopy VP3 was shown to be associated with apoptotic structures. In vitro,
expression of VP3 induced apoptosis in chicken lymphoblastoid T cells and myeloid cells, which are susceptible
to CAV infection, but not in chicken embryo fibroblasts, which are not susceptible to CAV. Expression of a
C-terminally truncated VP3 induced much less pronounced apoptosis in the chicken lymphoblastoid T cells.

Chicken anemia virus (CAV) transiently causes severe ane-
mia due to destruction of erythroblastoid cells and immuno-
deficiency due to depletion of cortical thymocytes in young
chickens (10, 38). Jeurissen et al. (11) have provided evidence
that the observed depletion of the thymocytes occurs via
CAV-induced apoptosis. Apoptosis is considered to be a
physiological process of cell depletion that is part of the
homeostatic regulation of normal tissues (5). CAV in addition
to several other viruses, such as human immunodeficiency virus
type 1 (2) and parvovirus B19 (19), seems to use apoptosis to
exert its cytopathogenic effect.
CAV is a small virus with a diameter of about 23 nm and
contains a circular single-stranded DNA of 2.3 kb (27). CAV
multiplies via a circular double-stranded DNA replicative
intermediate, which was recently cloned (18, 21). The cloned
CAV genome was proven to be representative for CAV
isolates collected worldwide (23, 29). A polycistronic polyade-
nylated mRNA (22) which comprises three overlapping open
reading frames encoding proteins VP1 (51.6 kDa), VP2 (24.0
kDa), and VP3 (13.6 kDa) is transcribed from the CAV
genome.
In the present paper, we report studies on the expression of
the VP3 protein in CAV-infected cells. VP3 is a putative CAV
protein of 121 amino acids and contains two proline-rich
stretches and two positively charged regions. So far, there is no
evidence that VP3 is present in purified CAV particles (27).
However, VP3 seems to be important for the virus life cycle, as
found by VP3-deletion studies (19a). By way of immunolabel-
ing experiments with CAV-infected chicken lymphoblastoid T
cells in vitro, we present evidence that VP3 accumulates in
condensed DNA. We also show that expression of VP3 alone
is sufficient to induce apoptosis in chicken mononuclear cells.
MATERIALS AND METHODS
Viruses and cells. The chicken lymphoblastoid T-cell line
MDCC-MSBI, transformed by Marek's disease virus (1), was
infected with the CAV-Cux-1 isolate, which was originally
*
Corresponding author. Mailing address: Laboratory for Molecular
Carcinogenesis, Sylvius Laboratory, Leiden University, P.O. Box 9003,
2300 RA Leiden, The Netherlands. Phone: (31) 71 276113. Fax: (31)
71 276125.
346
isolated in Germany (34). For DNA transfections, we used the
cell line MDCC-MSB1; the myeloid cell line LSCC-HD11
transformed by avian myeloblastosis virus (4), which was kindly
provided by T. Graf; and primary chicken embryo fibroblasts
(CEFs).
Immunoblotting. The nuclear protein preparations that
were used for immunoblotting were produced as follows.
MDCC-MSB1 cells that had been infected at a multiplicity of
infection of about 0.2 median tissue culture infective dose per
cell were collected by centrifugation at 48 h after infection, and
a nucleus-rich pellet was obtained (32). Nuclear material that
had been resuspended in 10 mM Tris HCl-1 mM EDTA (pH
8.0), dispersed by thorough sonication, and solubilized with 2%
sodium dodecyl sulfate (SDS) was fractionated by gel filtration
through a column (45.1 by 5 cm) containing Sephacryl S200.
Noninfected MDCC-MSB1 cells were similarly processed for
control purposes.
Proteins were separated by SDS-polyacrylamide gel electro-
phoresis, electroblotted, and subjected to immunodetection as
described by Todd et al. (28). Blots were incubated with a
1:10,000 dilution of an immunoglobulin fraction (approximate-
ly 28.7 mg/ml), which had been produced by precipitation with
(NH4)2SO4, of the CAV-specific monoclonal antibody (MAb)
3B1 (17) and a 1:1,000 dilution of a peroxidase-labeled goat
anti-mouse conjugate.
Immunoperoxidase assay and immunofluorescence assay.
MDCC-MSB1 cells, which were synchronized by growing in
medium without leucine and fetal bovine serum, were infected
with CAV at a multiplicity of infection of about 1 median tissue
culture infective dose of virus per cell. The cells were harvested
at 0, 16, 24, 32, and 64 h after infection; spun on coverslips; and
fixed with 100% acetone. Immunoperoxidase staining was
carried out with a 100-fold dilution of the MAb CVI-CAV-85.1
(85.1) (21) and a 500-fold dilution of a peroxidase-labeled
rabbit anti-mouse immunoglobulin G conjugate in phosphate-
buffered saline, as described by Jeurissen et al. (9). Immuno-
fluorescence assays were carried out on DNA-transfected cells
by using MAb CVI-CAV-85.1 as described by Noteborn et al.
(20).
Immunoelectron microscopy. MDCC-MSB1 cells were in-
fected with CAV at a multiplicity of infection of about 0.2
median tissue culture infective dose of virus per cell. The
infected and noninfected control cells were harvested 48 h
VOL. 68? 1994

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FIG. 1. Immunoblot of nuclear protein fractions from noninfected

(lane A) and CAV-infected (lane B) MDCC-MSBl cells. The blot was
initially allowed to react with MAb 3B1, which is directed against VP3.

after infection. Thin-section electron microscopy and immu- 64 64

nogold labeling were carried out as described by McNulty et al.
(16). The grids were stained with a 10,000-fold dilution of the
CAV-specific MAb 3B1 and a 1:50 dilution of 15-nm gold-
labeled goat anti-mouse immunoglobulin G conjugate.
Construction of VP3 expression vectors. Plasmid DNA
manipulations were performed essentially according to the
methods described by Maniatis et al. (14). The CAV sequences
originated from plasmid pCAV/E, described by Noteborn et al.
(21).
The sequences encoding VP3 were cloned in the expression
vector pRSV-H20, which contains the Rous sarcoma virus
promoter. From plasmid pEP-VP3 (19a) we isolated a 0.46-kb
BamHI-EcoRI DNA fragment with CAV DNA sequences
from positions 427 to 868 (21). It contains the coding region of
VP3, a 58-bp 5'-flanking sequence, and a 25-bp 3-flanking
sequence. Two synthetic DNA oligomers, 5'-GATCCAACCC
GGGTFG-3' and 5'-AATTCAACCCGGGTTG-3', were hy-
bridized to form a double-stranded BamHI-EcoRl DNA
linker. The vector pRSV-H20 was linearized with BglII and
treated with calf intestine alkaline phosphatase, after which a
4.3-kb fragment was isolated. The BamHI-EcoRI DNA linker
and the 0.46-kb BamHI-EcoRI DNA fragment were ligated
with the 4.3-kb BglII DNA fragment. The final construct,
pRSV-VP3, contains the VP3 coding region under the control
of the Rous sarcoma virus promoter as shown in Fig. 4 and was
analyzed by restriction enzyme digestions.
A truncated VP3 product was made by deleting 11 codons at
the 3' terminus of the VP3 coding sequences and introducing
a new stop codon. Plasmid pEP-VP3 was digested with BamHI
and HindIlI. The 0.38-kb BamHI-HindIIl DNA fragment was
isolated. Two synthetic DNA oligomers, 5'-AGCTTGATTAC
CACTACTCCCTGAG-3' and 5'-TCGACTCAGGGAGTAG
TGGTAATCA-3', were hybridized to form a double-stranded
D : .
FIG. 2. Indirect immunoperoxidase staining of CAV-infected
MDCC-MSB1 cells with antibody 85.1 directed against VP3. The cells
were fixed and stained 0, 16, 24, 32, 40, 56, and 64 h after infection.
HindIII-SalI DNA linker. Plasmid pRSV-H20 was digested
with BglII and Sall and treated with calf intestine alkaline
phosphatase, and a 4.3-kb fragment was isolated. The HindIll-
Sall DNA linker and the 0.38-kb BamHI-HindIII DNA frag-
ment were ligated with the 4.3-kb BglII-SalI DNA fragment.
The final construct, pRSV-tr, was analyzed by restriction
enzyme digestions and by sequencing of the newly introduced
bases (see Fig. 4).
DNA transfection and analysis of cellular DNA. Plasmid
DNA was purified by centrifugation in a CsCl gradient and by
column chromatography in Sephacryl S500 (Pharmacia). For
DNA transfections of MDCC-MSB1 and LSCC-HD11 cells,
the DEAE-dextran method of Luthman and Magnusson (13),
in which the chloroquine treatment was replaced with a
dimethyl sulfoxide boost, was used. CEFs were transfected
with DNA according to the calcium phosphate precipitation
method of Graham and Van der Eb (8). DNA extracted from
DNA-transfected MDCC-MSB1 cells was analyzed as de-
scribed by Jeurissen et al. (11).
348 NOTEBORN ET AL.
FIG. 3. Immunogold labeling of electron-dense nuclear inclusions
in CAV-infected MDCC-MSB1 cells. Infected cells harvested 48 h
after infection were embedded in Lowicryl and allowed to react with
MAb 3B1, which is directed against VP3. Magnification, x 26,000.
RESULTS
Expression of VP3 during CAV infection of cultured cells.
We have analyzed the expression of VP3 during a CAV
infection of chicken lymphoblastoid T cells. The CAV-infected
cells were analyzed by immunoblotting and an immunoperoxi-
dase assay using CAV-specific MAbs 3B1 (17) and 85.1 (21).
Both MAbs are specific for CAV VP3, since these specifically
bind to recombinant VP3 synthesized with a baculovirus
system (19a). Immunoblotting experiments showed that frac-
tions of the nuclear extracts of CAV-infected cells that eluted
close to the void volume of the Sephacryl S200 column
contained a 16-kDa protein that specifically reacted with MAb
3B1 (Fig. 1). Under the immunodetection conditions used,
faint nonspecific staining of other proteins in the profiles
derived from infected and noninfected cells was also observed.
Expression of sequences encoding VP3 in a baculovirus system
yields a protein with an identical molecular mass (19a).
Immunoperoxidase staining of CAV-infected MDCC-MSB1
cells with MAb 85.1 was carried out at several time points after
infection (Fig. 2). As early as 24 h after infection, VP3 was
present in the nucleus. The distribution of VP3 was very faint
and finely granular. Later after infection, the granules in-
creased in size, and gradually aggregates appeared. The aggre-
gates were especially prominent around 60 h after infection. At
this stage of infection, the CAV-specific cytopathogenic effect
was clearly visible.
The aggregates containing VP3 resemble the electron-dense
structures described by Jeurissen et al. (11) that occur during
the apoptotic process of CAV-infected cells. We have shown
by immunogold electron microscopy that MAb 3B1 binds
strongly to many, but not all, of the electron-dense apoptotic
J. VIROL.
A.
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B.
1-M N A L Q E D T P P G P S T V
F R P P T S S R P L E T P H C
R E I R I G I A G I T I T L S
L C G C A N A R A P T L R S A
T A D N S E S T G F K N V P D
L R T D Q P K P P S K K R S C
D P S E Y R V S E LK E S L I
T T T P S R P R T A K R R I R
L-1 21
FIG. 4. (A) Schematic representation of the expression vectors
pRSV-VP3 and pRSV-tr. Both vectors are constructed as described in
Materials and Methods. The CAV sequence encoding VP3 is drawn as
a filled box, that for VP3-tr is drawn as a striped box, the Rous sarcoma
virus promoter region is drawn as a dotted box, and the simian virus 40
sequences, containing a poly(A) addition site, is drawn as an open box.
(B) Amino acid sequence of VP3. The 11 amino acids which were
deleted from the C terminus of the truncated form of VP3 are
underlined. The proline residues are in italics, and the lysine and
arginine residues are in boldface type.
structures in the nucleus of CAV-infected cells (Fig. 3).
Apparently, VP3 accumulates in these apoptotic structures.
VP3 induces apoptosis in cultured avian mononuclear cells.
To establish whether VP3 alone is able to induce apoptosis, the
expression vector pRSV-VP3 was constructed (Fig. 4). This
vector contains CAV DNA sequences encoding only VP3.
After transfection with DNA of pRSV-VP3, VP3 was tran-
siently expressed in cultured chicken lymphoblastoid T cells.
The cells were harvested after 48 h, acetone fixed, and stained
with MAb 85.1. In addition, the cells were treated with
propidium iodide (PI), which is known to stain intact nuclei
strongly but apoptotic nuclei relatively weakly (26). CAV-
infected cells containing apoptotic inclusion structures, which
were comparable to the structures observed at 64 h after
infection (Fig. 2), were weakly and irregularly stained by PI
(data not shown). We observed 60% of the transfected cells
with nuclei exhibiting a very finely granular distribution of
VP3. The DNA of cells with this type of VP3 staining was
always clearly stained by PI. In contrast, 40% of VP3-positive
cells contained aggregates of VP3, and their DNA invariably
stained weakly and irregularly with PI. Three days after
transfection, almost all VP3-positive cells contain VP3 aggre-
gates and negatively PI-stained DNA (Fig. 5). In contrast,
expression of a C-terminally truncated VP3 (Fig. 4) yielded
almost no apoptosis in MDCC-MSB1 cells. Three days after
transfection, 80 to 90% of the cells expressing truncated VP3
VOL. 68, 1994
A B
C
E F
FIG. 5. Indirect immunofluorescence of MDCC-MSB1 cells trans-
fected with plasmid pRSV-VP3 (A to D) or pRSV-tr (E and F). The
cells were harvested 48 h (A and B) or 72 h (C to F) after transfection,
fixed, and stained either with MAb 85.1 directed against VP3 (A, C,
and E), or with PI (B, D, and F).
still have normally PI-stained nuclei (Fig. 5). At this time point,
the cellular DNA of MDCC-MSB1 cells expressing VP3
showed the apoptosis-specific nucleosomal laddering. This
laddering was proven to be much less in DNA from cells
transfected with plasmid pRSV-tr or absent in DNA from
MDCC-MSB1 cells transfected with plasmid pRSV-CAT,
which expressed the chloramphenicol transferase gene (Fig. 6).
We conclude that expression of VP3 alone can induce the
apoptotic process that is observed during CAV infection and
results in cell death.
To test whether VP3 can also induce apoptosis in other
chicken cell types, it was expressed in the myeloid cell line
LSCC-HD11 and in primary CEFs. LSCC-HD11 is susceptible
to CAV, whereas CEFs are not. Transfection of DNA encod-
ing VP3 in LSCC-HD1 1 cells yielded the formation of VP3
aggregates and weak and irregular staining of the cellular DNA
by PI. CEFs expressing VP3 revealed only granular staining of
VP3 and strong staining with PI (Fig. 7). This suggests that
VP3 can induce apoptosis in LSCC-HD 11 cells but not in
CEFs, or at least to a much lesser extent.
APOPTOSIS INDUCED BY A SINGLE CAV PROTEIN 349
m 1 2 3 4
slot-
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FIG. 6. DNA fragmentation in MDCC-MSBI cells undergoing
VP3-mediated cell death. DNAs from CAV-infected MDCC-MSBI
cells which were harvested 72 h after infection (lane 1) and from
MDCC-MSB1 cells transfected with DNA of plasmids pRSV-VP3
(lane 2), pRSV-tr (lane 3), and pRSV-CAT (lane 4) were analyzed on
a 1% agarose-ethidium bromide gel. Lane m, DNA markers (lambda-
HindIll fragments; Promega). About 10% of the MDCC-MSBI cells
expressed VP3 or VP3-tr protein, as determined by immunofluores-
cence (data not shown).
DISCUSSION
CAV induces cell death of chicken lymphoblastoid T cells
via apoptosis (11). By use of immunoassays and DNA analysis,
we have established that the putative open reading frame for
VP3 of the CAV genome (18, 21) is indeed expressed in
CAV-infected cells. Early after infection, VP3 is colocalized
with cellular DNA. Later after infection, VP3 is bound to
nuclear aggregates that are known to be apoptotic bodies (5,
1 1). Expression of only VP3 in MDCC-MSB1 cells mimics the
process of CAV-induced apoptosis. In general, apoptosis is a
process leading to cell death that occurs rapidly (36). The
majority of CAV-infected MDCC-MSB1 cells become apop-
totic within 64 h after infection. In vitro expression of VP3 in
transfected cells causes CAV-like apoptosis within 3 days.
These data suggest that VP3 alone can trigger the apoptotic
pathway in CAV-infected cells.
How does the expression of VP3 cause apoptosis? In
general, apoptosis can be induced by a variety of viral and
nonviral external stimuli, which can converge on a common
intracellular pathway, resulting in the activation of an endonu-
clease (15). The amino acid sequence of VP3 does not show
any distinct homology with the sequences of the receptors that
mediate apoptosis (6, 24, 25, 35) or of c-myc and p53 oncogene
products (7, 37), known to induce apoptosis. So, it is unlikely
that VP3 mimics the activity of one of these cellular proteins.
Further experiments have to be carried out to prove whether
VP3 causes apoptosis by inhibition of the antiapoptosis activity
of the oncogene product Bcl-2 (33). We also cannot exclude
the possibility that VP3 induces apoptosis by binding to one of
350 J. VIROL.
NOTEBORN ET AL.J.Vto.

densation of the cellular DNA. A disturbance of the supercoil-

A B ing organization by VP3 might be caused by its high proline
content. Induction of apoptosis by disruption of the superchro-
matin structure was described for apoptotic rat thymocytes
(31) and rat ventral prostate cells after castration (12). An
alternative explanation might be that VP3 interacts directly
with the histone protein HI, which binds to the intranucleo-
somal DNA. For instance, binding of VP3 to this region
instead of histone HI might enable endonucleases to digest the
intranucleosomal DNA. Arends et al. (3) have observed that
the oligonucleosomes become depleted of histone HI during
the apoptotic process.
Under our experimental conditions, the expression of VP3
in CEFs and cells of the human epithelia] cell line HeLa (data
not shown) did not result in the observed disruption of the

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cellular DNA. However, neither cell line is susceptible to
CAy, in contrast to MDCC-MSBi and HDll cells. It might
well be that VP3 can cause degradation of cellular DNA only
in cells that are susceptible to CAy. An alternative explanation
might be that VP3 induces a cascade of events, resulting in
single-strand modification, i.e., the generation of alkaline-
sensitive sites in the cellular DNA. Apparently, in lymphocytes
E F and monocytes these modifications lead to double-stranded
cleavage, whereas HeLa cells and CEFs remain arrested at the
stage of single-strand modification. This would account for the
observation that lymphocytes and monocytes fragment their
DNA within hours after treatment with VP3. This explanation
was offered by Tomei (30) for the glucocorticoid-induced
apoptosis observed in lymphocytes but not in fibroblasts and
HeLa cells.
We conclude that a single CAV protein can induce apoptosis
in chicken lymphoblastoid T and myeloid cells. Experiments
that will unravel the mechanism by which VP3 induces cell
death are under way.

ACKNOWLEDGMENTS
FIG. 7. Indirect immunofluorescence of LSCC-HDI I cells and
We thank Hans van Ormondt for critically reading the manuscript,
CEFs which were transfected with plasmid pRSV-VP3, which encodes
VP3. The LSCC-HDII
Alt Zantema for helpful discussions, and Frans van Bussel for his
cells (A to D) were harvested 48 h after
transfection, and the CEFs
excellent drawing work.
(E and F) were harvested 72 h after
transfection. The cells fixed and stained with MAb 85.1 directed
This research was made possible partially with research grants from
were
The Netherlands Ministry of Economy Affairs and Aesculaap By,
against VP3 (A, C, and E) and with PI (B3, D, and F).
Boxtel, The Netherlands.
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