JOURNAL OF VIROLOGY, Apr. 1998, p. 2697–2707 Vol. 72, No.

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0022-538X/98/$04.0010
Copyright © 1998, American Society for Microbiology

Separate DNA Elements Containing ATF/CREB and IE86

Binding Sites Differentially Regulate the Human
Cytomegalovirus UL112-113 Promoter at Early
and Late Times in the Infection
STEVEN M. RODEMS, CHARLES L. CLARK, AND DEBORAH H. SPECTOR*
Department of Biology and Center for Molecular Genetics, University of California, San Diego,
La Jolla, California 92093-0357
Received 18 November 1997/Accepted 23 December 1997

The human cytomegalovirus (HCMV) UL112-113 promoter represents a useful model for studying temporal
regulation of viral gene expression. Stimulation of this promoter by the 86-kDa immediate-early protein (IE86)
is controlled by sequences between nucleotides 2113 and 259, which include both an ATF/CREB and an IE86
binding site. In transient assays, the ATF/CREB site is essential, and the IE86 site, although nonessential, can
enhance transcription. With recombinant viruses, we have assessed the function of these promoter elements in
the context of the viral genome. Transcription from the inserted UL112-113 promoter shows the same temporal
pattern as the endogenous promoter, including the switch to an upstream RNA start site late in infection.
Deletion of sequences containing the IE86 site results in a decrease in the level of early transcription and
elimination of late transcription. In contrast, when the ATF/CREB site is deleted, early RNA synthesis is
almost completely abolished, but late transcription is comparable to that of the wild type, with repositioning
of the RNA start site downstream by the number of nucleotides deleted. Replacement of sequences between
2108 and 295 with the HCMV cis-repression signal from the major immediate-early promoter had no effect
on the level of late RNAs but resulted in the repositioning of the RNA start site 39 nucleotides upstream. These
results suggest that the ATF/CREB site is functional only at early times, while sequences containing the IE86
site modulate the level of early RNAs and may be required for activating late transcription in a distance-
dependent manner.

Human cytomegalovirus (HCMV), a member of the beta-
herpesvirus family, is an important opportunistic pathogen in
immunocompromised individuals and is recognized as a major
viral cause of birth defects in newborns (2). As for other her-
pesviruses, HCMV genes are expressed in a temporal pattern
upon infection (5, 18, 29, 34, 35). Members of the immediate-
early (IE) class of genes are expressed upon viral infection and
do not require de novo protein synthesis for production of
their RNAs. Several of the IE proteins are transactivators of
the products of the next class of genes, the early genes. Con-
comitant with viral DNA replication, there is expression of the
late class of viral genes, whose protein products are generally
involved in viral genome packaging and virion maturation.
Since several of the early gene protein products are essential
for viral DNA replication, understanding the control of early
viral gene expression is important for designing therapeutic
strategies to inhibit viral replication.
In order to understand the mechanisms that control early
viral gene expression, several laboratories have studied a num-
ber of different early viral promoters by various types of tran-
scription assays (for reviews, see references 20 and 26). To
date, the most common assay to study viral promoter activity
has been the transient expression assay. In this assay, a plasmid
containing a reporter gene under control of an early viral
promoter is transfected into cells along with expression plas-
* Corresponding author. Mailing address: Department of Biology,
0357, University of California, San Diego, 9500 Gilman Dr., La Jolla,
CA 92093-0357. Phone: (619) 534-9737. Fax: (619) 534-6083. E-mail:
dspector@ucsd.edu.
mids encoding HCMV transcriptional regulatory proteins.
These assays have proven useful in determining which pro-
moter sequences are important for gene expression as well as
which HCMV proteins behave as transcriptional regulators. In
fact, these assays have identified the HCMV IE2 86-kDa pro-
tein (IE86) and the IE1 72-kDa protein (IE72) as the major
transregulators of early viral gene expression (for reviews, see
references 20 and 26). However, transient expression assays
are limited in that they do not provide accurate information
about the regulatory events that occur during a normal viral
infection. Several labs have attempted to circumvent this issue
by transfecting cells with plasmid and subsequently superin-
fecting with virus (3, 10, 25, 27, 32). Although this allows one
to study gene expression in infected cells, there remain tem-
plate-specific differences between plasmid DNA and viral
DNA. In particular, the late induction of the 1.2-kb RNA
promoter, normally observed in infected cells, does not occur
when the promoter is located on a plasmid in transfection-
infection assays unless an HCMV origin of replication, oriLyt,
is also included on the plasmid (33). In addition, it was shown
that at least two genes, UL83 and UL99, with early-late and
true late kinetics, respectively, were activated earlier and to
higher levels in transfection-infection assays than observed
with the endogenous genes during a normal infection (14). In
an attempt to study early and late viral gene expression without
the inaccuracies associated with transfection assays, Kohler et
al. have used a gene replacement strategy based on the finding
that a portion of the unique short region of the HCMV ge-
nome is dispensable for viral growth (14). To this end, they
have constructed recombinant viruses in which a viral pro-
moter linked to a reporter gene is inserted between the US9

2697
2698 RODEMS ET AL.
and US10 genes and have shown that the inserted promoters
exhibit kinetics similar to those of the endogenous viral pro-
moters.
As a model for early HCMV gene expression, our laboratory
has studied the expression of the 2.2-kb class of RNAs (open
reading frame UL112-113) which encode a family of nuclear
phosphoproteins (13, 27, 28, 36, 37). Although the function of
these proteins is unclear, there is evidence that they behave as
transcriptional activators and are required for viral DNA rep-
lication (7, 10, 21). By transient expression analysis, our labo-
ratory has shown that activation of the UL112-113 promoter is
mediated through a major regulatory domain between nucle-
otides 2113 and 259 (23, 24). This region contains a weak
binding site for the HCMV transactivator IE86 (2113 to 285)
and a consensus ATF/CREB binding site (271 to 266) (15, 23,
24). In addition, the UL112-113 promoter contains three other
binding sites for IE86 (2286 to 2257, 2248 to 2218, and
2148 to 2120) (1, 24). Results of mutational analyses of the
UL112-113 promoter suggest that in transfection assays, the
ATF/CREB site is the major regulatory element upstream of
the TATA box, whereas the IE86 binding sites play an acces-
sory role (1, 15, 23, 24).
The goal of this study was to determine the functions of
various sequence elements within the UL112-113 promoter at
different times during HCMV infection. To this end, we have
further defined the roles of the sequences containing the ATF/
CREB binding site and the weak IE86 binding site within the
UL112-113 promoter at early and late times during infection.
We have constructed recombinant viruses in which various
mutations of the UL112-113 promoter, linked to the chloram-
phenicol acetyltransferase (CAT) gene, were inserted between
the US9 and US10 genes in the viral genome and have ana-
lyzed UL112-113 promoter–CAT activity at different times
during the infection. We find that the kinetics of expression of
the UL112-113 promoter from the US9/10 locus are identical
to that of the endogenous viral promoter, including the switch
to a different RNA start site late in infection (28). Consistent
with transient expression data, our results demonstrate that the
sequences containing the ATF/CREB site play a major role in
UL112-113 promoter activity early during viral infection,
whereas the region containing the weak IE86 binding site has
a moderate effect on transcription. However, at late times in
the infection, we find that the ATF/CREB site plays little if any
role in expression, but the sequences between 2113 and 285,
which include the weak IE86 binding site, are required for
transcription from the late RNA start site within the UL112-
113 promoter. Furthermore, replacement of the region be-
tween nucleotides 2108 and 295 with the HCMV cis-repres-
sion signal (CRS) indicated that the sequences between 2108
and 295 play a role in distance-dependent, and possibly ori-
entation-dependent, late transcription initiation from the
UL112-113 promoter. These data underscore the value of us-
ing recombinant viruses to study viral promoter activity and
demonstrate that different promoter regulatory elements are
employed at early and late times in the infection.
MATERIALS AND METHODS
Cells and virus. Human foreskin fibroblasts (FF cells) were maintained in
minimum essential medium with Earle’s salts containing 10% fetal bovine serum.
HCMV Towne strain was obtained from American Type Culture Collection.
Methods for cell culture and viral infection have been described elsewhere (30).
All infections were performed with a multiplicity of infection of 3 to 10.
Molecular cloning. Sequences used to facilitate homologous recombination
into the HCMV US9/10 locus were derived from a 3.4-kb EcoRI-HindIII frag-
ment from pHCMV-EcoRI-B (30). The 3.4-kb fragment was cut with SalI and
end repaired with Klenow enzyme, and ligated to HindIII linkers, and the
resulting 2.7-kb fragment was ligated into pGem-3Z at HindIII to give pGem-
J. VIROL.
US9/10. Two oligonucleotides (59-CAGATCTGCGGCCGCAGGCC-39 and 59-
TGCGGCCGCAGATCTGGGCC-39) were annealed, resulting in a 20-bp ApaI
fragment containing internal BglII and NotI sites which was subsequently ligated
into the ApaI site of pGem-US9/10. The resulting plasmid was cut with BglII, and
a 4.8-kb BamHI fragment from pON855 (31), containing the lacZ and guanine
phosphoribosyltransferase (gpt) genes, was inserted to give rise to pUS9/10-lacZ/
gpt.
A 1.95-kb BamHI fragment from p358-CAT (27) was end repaired with Kle-
now enzyme, ligated to NotI linkers, and inserted at the NotI site of pUS9/10-
lacZ/gpt to give pUS9/10-358-CAT. p148-CAT (27), p119-CAT (previously re-
ferred to as D [23]), and p148(D84-58)-CAT (previously referred to as Del [23])
were digested with HindIII and BamHI, end repaired with Klenow enzyme,
ligated to NotI linkers, and inserted at the NotI site of pUS9/10-lacZ/gpt to give
pUS9/10-148-CAT, pUS9/10-119-CAT, and pUS9/10-148(D84-58)-CAT, respec-
tively.
p148(D84-58)CRS-CAT and p148(D84-58)IICRS-CAT were constructed by
using a QuickChange site-directed mutagenesis kit (Stratagene). p148(D84-
58)CRS-CAT was constructed according to the manufacturer’s protocol, using
the two oligonucleotides 59-CTAGAGTACCAGTCGTTTAGTGAACCGTAC
TGTTTAAGGG-39 and 59-CCCTTAAACAGTACGGTTCACTAAACGACT
GGTACTCTAG-39 and the plasmid p148(D84-58)-CAT. p148(D84-58)IICRS-
CAT was constructed similarly, using the two oligonucleotides 59-GTTTAGTG
AACCGTTTAGTGAACGGGTGTTGCTAGG-39 and 59-CCTAGCAACACC
CGTTCACTAAACGGTTCACTAAAC-39 and the plasmid p148(D84-58)CRS-
CAT. HindIII-BamHI fragments from p148(D84-58)CRS-CAT and p148(D84-
58)IICRS-CAT were end repaired with Klenow enzyme, ligated to NotI linkers,
and inserted into the NotI site of pUS9/10-lacZ/gpt to give pUS9/10-148(D84-
58)CRS-CAT and pUS9/10-148(D84-58)IICRS-CAT, respectively.
Recombinant virus construction. All plasmids derived from pUS9/10-lacZ/gpt
were linearized with HindIII, and 30 mg was electroporated into 4 3 106 FF cells.
Twenty-four hours after electroporation, the cells were infected with wild-type
HCMV (Towne strain) at a multiplicity of infection of 5 to 10. Infections were
allowed to proceed for 5 days, and virus was harvested by clarifying the culture
supernatant at 1,500 rpm for 5 min. Virus was passaged three times under
selection as follows. Briefly, cells were infected, and the inoculum was removed
3 h later and replaced with media containing 150 mM mycophenolic acid and 10
mM xanthine. After 5 days, the supernatant was collected, the cells were soni-
cated to release membrane-associated virus, and the supernatants were com-
bined. Virus was then added to fresh cells, and plaques were stained with
5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal; 0.5 mg/ml) to visual-
ize recombinants. Recombinant viruses were subsequently plaque purified two to
three times. The purified virus was then used to infect 5 3 105 cells. The viral
supernatant was used to further amplify the virus, and genomic DNA was har-
vested from the cells to verify virus genotype by Southern blot analysis.
Southern blot analysis. Genomic DNA from mock or virus-infected cells was
isolated by using a QIAamp blood kit (Qiagen) as described in the manufactur-
er’s protocol. To confirm the presence of the CAT gene in the recombinant
viruses, genomic DNA was digested with NotI and subjected to Southern blot
analysis by using standard methods. Blots were hybridized to a 32P-labeled,
551-bp HindIII-to-NcoI DNA fragment isolated from pSV2-CAT (American
Type Culture Collection) and subjected to autoradiography. To test for purity of
recombinant viruses and to confirm insertion into the US9/10 locus, genomic
DNA was digested with EcoRI and NcoI, and Southern blot analysis was per-
formed with a 32P-labeled, 4.5-kb EcoRI-to-NcoI DNA fragment isolated from
pHCMV-EcoRI-B.
Western blot analysis. FF cells were infected with HCMV recombinants and
harvested at the indicated times by lysing directly in Laemmli sample buffer.
Lysates (104 cell equivalents) were electrophoresed on 10% polyacrylamide
protein gels and transferred to Immobilon membranes (Millipore). Blocked
membranes were incubated with primary antibody BSA 2-9 (37), followed by
incubation with a horseradish peroxidase-coupled secondary antibody and de-
tection with chemiluminescence (Pierce) according to standard methods.
CAT enzyme assays and RNA primer extension analysis. FF cells were in-
fected with HCMV recombinants, harvested at the indicated times, and assayed
for CAT enzyme as previously described (27). CAT assays were quantitated by
scintillation counting or phosphorimager analysis.
For primer extension analysis, 7 3 106 FF cells were infected with recombinant
HCMV and harvested at the indicated times by trypsinization. Total RNA was
isolated from cell pellets by using a Qiagen RNeasy Midi kit. Primer extension
reactions were performed as described previously by using a 32P-end-labeled
oligonucleotide complementary to CAT RNA (positions 115 to 153 relative to
the ATG codon) and 50 mg of total RNA (22).
RESULTS
Construction of recombinant HCMV. The HCMV UL112-
113 promoter has been extensively studied by using plasmid
transfections, in vitro transcription, and DNase I footprint
analyses (1, 13, 15, 23, 24, 27, 28, 36). However, these assays
are limited in that certain aspects of infection-specific viral
VOL. 72, 1998 HCMV UL112-113 PROMOTER REGULATION DURING INFECTION 2699

FIG. 1. UL112-113 promoters inserted into recombinant viruses. (A) Sequences of the HCMV UL112-113 promoter from 2113 to 11. Previously identified
regulatory elements are indicated by the shaded boxes beneath the sequence. The IE86 binding site represents the sequences protected by IE86 in a DNase I footprint
analysis (24), whereas the CREB/ATF and TATA sites are the respective consensus sequences. (B) Map of UL112-113 promoter mutations used in this study. The name
of each recombinant virus is indicated at the right. The weak IE86 binding site, CREB/ATF site, and TATA box are indicated by shaded boxes. The black box indicates
the replacement of the weak IE86 binding site and/or adjacent A-T rich sequences with the HCMV CRS from the major IE promoter. Dashed lines denote deleted
sequences. (C) Sequence between 2113 and 285 in v148(D84-58)-CAT, v148(D84-58)CRS-CAT, and v148(D84-58)IICRS-CAT. Sequences protected by IE86 in a
DNase I footprint are indicated by the shaded box above the sequence. Sequences outlined in black are those replaced in v148(D84-58)CRS-CAT and v148(D84-
58)IICRS-CAT. The CRS is indicated by the bracket.

gene regulation are absent. In particular, late viral transcrip-
tion is inaccurately represented in plasmid transfections (14).
To better understand the regulation of the UL112-113 pro-
moter during infection, we constructed recombinant HCMV in
which the UL112-113 promoter, linked to the CAT gene, was
inserted into the viral genome. The recombinant viruses were
then used to analyze the role of various UL112-113 promoter
elements throughout the infection.
The region containing the US1 to the US11 genes has been
shown to be dispensable for viral growth (8, 9). Stable recom-
binant HCMV can be constructed by insertion of sequences
between the US9 and US10 genes through homologous recom-
bination (14). Therefore, we have used this technique to insert
the UL112-113 promoter–CAT sequences into the viral ge-
nome. We constructed several different recombinant viruses
containing previously uncharacterized site-directed mutations
as well as promoter mutations that have been previously ana-
lyzed by transient expression analysis (Fig. 1). Recombinant
viruses were propagated under GPT selection, and putative
recombinants were identified by X-Gal staining as described in
Materials and Methods.
The genotype of the recombinant viruses was confirmed by
Southern blot analysis (Fig. 2). To confirm the presence of
CAT sequences, genomic DNA preparations from infected
cells were digested with NotI, which should yield a fragment
containing the CAT gene and the UL112-113 promoter. The
size of this fragment varies from 1.8 to 2.1 kb, depending on
the promoter mutation inserted. Southern blots were probed
with a 32P-labeled, 551-bp HindIII-NcoI DNA fragment con-
taining only CAT sequences. As expected, CAT sequences
were not detected in wild-type- or mock-infected cells (Fig. 2C,
lanes 1 and 2). However, a single specific band between 1.8 and
2.1 kb was detected in all recombinant viruses, confirming the
presence of the CAT gene (Fig. 2C, lanes 3 to 7). Furthermore,
the size of each band was consistent with the length of the
UL112-113 promoter mutation inserted.
To test for purity of recombinant viruses and to confirm
proper insertion into the US9/10 locus, genomic DNA was
digested with EcoRI and NcoI, which, in wild-type virus, yields
a 4.5-kb fragment spanning the US9-US12 genes. This 4.5-kb
sequence was also used as a probe for Southern blots. The
4.5-kb fragment was detected in cells infected with wild-type
virus, whereas no signal was detected in mock-infected cells
(Fig. 2D, lanes 1 and 2). In recombinant viruses, an EcoRI/
NcoI digest gave a pattern different from that of wild-type virus
due to the presence of two additional EcoRI sites within the
inserted sequences (Fig. 2B). Therefore, the 4.5-kb probe de-
tected two fragments flanking the insertion site; a 2.5-kb frag-
ment containing the US9 and gpt genes, and a fragment con-
taining the US10-12 genes, the UL112-113 promoter, and CAT
sequences (Fig. 2D, lanes 3 to 7). The size of the latter frag-
ment varied from 3.3 to 3.6 kb, depending on the promoter
mutation. The pattern shown in Fig. 2D confirmed the site of
insertion between the US9 and US10 genes. The purity of the
recombinants was verified by the absence of a 4.5-kb band on
longer exposures (data not shown).
Analysis of viral protein production in recombinant virus
infection. The US9/10 region has been shown to accept the
2700 RODEMS ET AL. J. VIROL.

FIG. 2. Site of recombination in the HCMV genome and Southern blot analysis of recombinant viruses. (A) Schematic representation of the site of recombination
within the HCMV genome. Shown is the HCMV Towne strain. White boxes denote the terminal repeat sequences. Black bars beneath the genome indicate the location
of the endogenous UL112-113 genes and the site of insertion within the region between the US9 and US12 genes. (B) Expanded map of the region between the US9
and US12 genes in recombinant HCMV. Where indicated, the direction of the open reading frame is shown with an arrow. Sizes of fragments expected to hybridize
to the CAT probe in Southern blots are shown above the map; sizes of fragments expected to hybridize to the genomic probe are shown below the map. Angled lines
between the shaded bars of the genomic probe indicate that the probe is contiguous. (C) Southern blot of genomic DNA from mock- and virus-infected cells, using
the CAT probe for detection. Viruses analyzed are indicated above the autoradiogram. The expected size range of the hybridized DNA fragment is indicated on the
right. Positions of molecular weight markers are indicated on the left. W.T., wild type. (D) Southern blot of genomic DNA from mock- and virus-infected cells, using
the genomic probe for detection. Viruses analyzed are indicated above the autoradiogram. The expected size ranges of the hybridized DNA fragments are indicated
on the right. Positions of molecular weight markers are indicated on the left.

insertion of exogenous DNA without affecting viral growth (11,
14). However, it was important to verify that all constructed
recombinant viruses had normal growth characteristics. To as-
sess the growth characteristics of the recombinant viruses and
to confirm that the endogenous UL112-113 promoter behaved
normally, we analyzed the kinetics of expression of the endog-
enous UL112-113 family of proteins during recombinant virus
infection. In a wild-type virus infection, a 43-kDa protein is
detected by 8 h after infection; by 48 to 72 h after infection, the
34-, 50-, and 84-kDa proteins are also detected (37). By West-
ern blot analysis, we determined that the pattern of expression
of the UL112-113 family of proteins in the recombinant viruses
was similar to that in wild-type Towne virus at both early and
late time points (Fig. 3). In addition, all recombinant viruses
grew to titers similar to that of wild-type Towne virus (data not
shown). These results suggest that insertion of sequences be-
tween the US9 and US10 genes had no effect on viral growth
or endogenous UL112-113 promoter activity.
Analysis of CAT protein and RNA expression from the
US9/10 locus in the recombinant virus v358-CAT. The recom-
binant virus designated v358-CAT contains wild-type UL112-
113 promoter sequences from 2323 to 135 (Fig. 4A). These
promoter sequences contain four binding sites for the HCMV
transactivator protein IE86 and one ATF/CREB binding site
upstream of the TATA box (1, 15, 23, 24). v358-CAT was used
to test the pattern of expression of the UL112-113 promoter
when inserted into the US9/10 locus. CAT assays performed on
lysates harvested at 8, 24, 48, and 72 h after infection showed
low levels of protein at 8 h after infection followed by an
accumulation of higher levels of protein starting at 24 h after
infection (Fig. 4B). This was similar to the accumulation of
protein levels of the endogenous UL112-113 family of proteins
at the same times after wild-type virus infection (Fig. 3) (37).
UL112-113 promoter expression from the US9/10 locus was
also examined by primer extension analysis using a primer that
hybridizes to sequences within the CAT gene (Fig. 4C). By
using total RNA isolated at 8, 24, 48, and 72 h after infection,
it was demonstrated that the previously mapped early mRNA
start site for the UL112-113 promoter (28) was also used when
the promoter was inserted in the US9/10 region. Beginning at
48 h after infection, transcription from the early (11) start site
declined and transcription initiated from a new start site at
VOL. 72, 1998 HCMV UL112-113 PROMOTER REGULATION DURING INFECTION 2701

FIG. 3. Western blot analysis of the UL112-113 proteins during infection with recombinant viruses. Western blot analysis was performed with total protein isolated
from infected cells at 8 (A), 24 (B), 48 (C), and 72 (D) h after infection as described in Materials and Methods. The recombinant viruses used are indicated above each
blot. Sizes of the UL112-113 proteins are indicated in kilodaltons on the right of each blot; positions of molecular weight markers are indicated in kilodaltons on the
left of each blot. The band detected just above 66 kD appears to be a cross-reactive protein and has been previously observed in some experiments (36). The band near
116 kDa is detected only with recombinant viruses and may represent LacZ expressed from the human b-actin promoter. wt, wild type.

262. These kinetics, including the switch in transcriptional
start sites at late times, mimic that of the endogenous UL112-
113 promoter (28), demonstrating that insertion of promoter
sequences within the US9/10 locus represents a valid method
to study UL112-113 promoter activity in the context of a viral
infection.
CAT activity from UL112-113 recombinant viruses. Previous
transient expression assays have shown that the three IE86
binding sites between 2323 and 2113 have very little effect on
UL112-113 promoter activity (1, 15, 23, 24). These experiments
also identified the ATF/CREB site between 271 and 266 as
the major element upstream of the TATA box contributing to
promoter activity. Therefore, in determining which UL112-113
promoter sequences are important during viral infection, we
have concentrated on the sequences between 2113 and 135,
which contain, in addition to the ATF/CREB site and the
TATA box, a weak IE86 binding site (Fig. 1A) (24). The
recombinant virus containing the sequences between 2113 and
135 is designated v148-CAT (Fig. 1B). CAT assays performed
with lysates harvested at 8, 24, 48, and 72 h after infection with
v148-CAT showed kinetics similar to that of v358-CAT (com-
pare Fig. 5 to Fig. 4B). CAT levels at each time point were
about twofold lower in v148-CAT infections compared to v358-
CAT, confirming that the three upstream IE86 binding sites in
combination have only a moderate effect on promoter activity.
Our initial analysis also included two recombinant viruses
containing promoter deletions that were previously analyzed in
transient expression assays (23). Recombinant virus v119-CAT
contains sequences from 284 to 135 and is missing all IE86
binding sites but maintains the ATF/CREB site and TATA box
(referred to as D in reference 23). Recombinant virus
v148(D84-58)-CAT is deleted for sequences between 284 and
258 and contains the weak IE86 binding site and the TATA
box but lacks the ATF/CREB site (referred to as Del in ref-
erence 23).
In transient assays, it was previously shown that deletion of
the weak IE86 binding site resulted in a twofold drop in pro-
moter activity (23). Analysis of CAT activity derived from
v119-CAT infection showed a similar twofold drop in CAT
activity relative to v148-CAT when assayed at 8 and 24 h after
infection. However, at 48 and 72 h after infection, there were
decreases of 6.8- and 15.8-fold, respectively (Fig. 5). These
2702 RODEMS ET AL.
FIG. 4. Analysis of CAT RNA and protein expression during infection with
v358-CAT recombinant virus. (A) Map of the UL112-113 promoter and CAT
gene inserted into v358-CAT. Sequences from 2323 to 135 were linked to the
CAT gene and inserted into a recombinant virus as described in Materials and
Methods. Arrows indicate the location and direction of transcription of RNA
from the early start site (11) and the late start site (262). Previously identified
regulatory regions are indicated by the shaded boxes. (B) CAT activity detected
in lysates from v358-CAT-infected cells. Protein lysates from infected cells were
harvested at 8, 24, 48, and 72 h after infection with v358-CAT. CAT activity was
determined as described in Materials and Methods and is represented as percent
acetylation per microgram of lysate used in the reaction. Time points are indi-
cated below the histogram. (C) Primer extension analysis of total RNA isolated
from v358-CAT-infected cells. Total RNA was isolated from infected cells at 8,
24, 48, and 72 h after infection, and primer extension analysis was performed as
described in Materials and Methods, using a primer which detects CAT mRNA.
Extension products were separated by denaturing polyacrylamide gel electro-
phoresis and subjected to autoradiography. Products were electrophoresed ad-
jacent to a sequencing ladder (lanes T, G, C, and A) generated with plasmid
p148-CAT and the identical CAT primer to identify transcriptional start sites.
Locations of start sites are indicated at the right. Time points are indicated below
the autoradiogram. hpi, hours postinfection.
data suggested that although sequences containing the IE86
binding site play a modest role early infection, those sequences
play a greater role in UL112-113 promoter activity late in
infection.
It was also shown by transient assays that deletion of the
ATF/CREB site results in a 20-fold drop in UL112-113 pro-
moter activity (23). Analysis of CAT activity derived from
v148(D84-58)-CAT infection showed a similar dependence on
the ATF/CREB site early during infection. At 8, 24, and 48 h
after infection, deletion of the ATF/CREB site resulted in
19.6-, 51.2-, and 68.9-fold decreases in CAT activity, respec-
tively, relative to v148-CAT. However, between 48 and 72 h
after infection with v148(D84-58)-CAT, CAT levels were stim-
ulated approximately 52.8-fold. Furthermore, CAT levels 72 h
after infection with v148(D84-58)-CAT were only twofold
lower than that detected 72 h after infection with v148-CAT.
Although the ATF/CREB site is a major regulatory element
early in the infection, these data suggest that this site plays
little if any role at late times in the infection. Taken together,
these results demonstrate that separate DNA elements differ-
entially regulate the UL112-113 promoter at early and late
times in the infection.
Primer extension analysis of UL112-113 recombinant vi-
ruses. Since the ATF/CREB site and sequences containing the
weak IE86 binding site seem to have different effects on pro-
moter activity depending on the stage of the infection, we
J. VIROL.
sought to determine what role these sequences played in the
initiation of transcription from the early (11) and late (262)
RNA start sites. To this end, primer extension analysis was
performed on RNA isolated at 8, 24, 48, and 72 h after infec-
tion with v148-CAT, v119-CAT, and v148(D84-58)-CAT (Fig.
6). During infection with v148-CAT, transcription initiation
from the 11 site increased up to 24 h after infection and then
declined by 48 and 72 h (Fig. 6A). As with v358-CAT, a shift in
the transcriptional start site to 262 was first observed at 48 h
after infection and increased between 48 and 72 h. The levels
of extended product mapping to 11 were slightly lower with
v148-CAT than with v358-CAT. However, the levels mapping
to 262 were almost identical, suggesting that the IE86 binding
sites upstream of 2113 have no effect on transcription from the
late, 262 mRNA initiation site.
CAT activity observed during infection with v119-CAT sug-
gested that the sequences from 2113 to 285, containing the
IE86 binding site, had little effect on promoter activity early
during infection but had a more dramatic role late. By primer
extension analysis of RNA from v119-CAT infections, we
found that transcription from the 11 site was identical to that
seen with v148-CAT (Fig. 6B). However, mRNA initiation
from 262 was completely abolished in v119-CAT infections.
Since the 262 initiation site is still present in v119-CAT, these
results suggested that sequences upstream from 262, which
included the IE86 binding site, were important in directing the
initiation of transcription late in infection.
Primer extension analysis was also performed with RNA
isolated from v148(D84-58)-CAT infections. Deletion of the
ATF/CREB site (which also deleted the 262 position) inhib-
ited transcription from the 11 site, providing further evidence
for the role of the ATF/CREB site in early UL112-113 pro-
moter activity. The deletion of promoter sequences from 284
to 258 placed the sequences containing the IE86 binding site
26 nucleotides closer to the TATA box. In turn, it was observed
that the initiation site detected at 48 and 72 h after v148-(D84-
58)-CAT infection was shifted 28 nucleotides downstream of
the original 262 position to the 234 position (Fig. 6C, lanes 11
and 12). Taken together, these data suggest that the site of
transcription initiation from the UL112-113 promoter late in
infection is determined not by the sequence of the initiation
site but by upstream sequences, which contain the IE86 bind-
ing site, in a distance-dependent manner. Furthermore, since
the level of extended product detected at 48 and 72 h after
infection with v148(D84-58)-CAT was comparable to that de-
tected after v148-CAT infection (compare Fig. 6C, lanes 11
and 12 with Fig. 6A, lanes 3 and 4), the ATF/CREB binding
site was not required for late transcription from the UL112-113
promoter.
Late transcription initiation from the UL112-113 promoter.
The UL112-113 promoter sequences between 2113 and 285,
which seem to be responsible for distance-dependent initiation
of transcription late in infection, contain a weak IE86 binding
site. In addition, two A-T-rich sequences are present within
this region (Fig. 1C). To determine which sequences within
2113 to 285 were involved in directing late transcription ini-
tiation, we constructed two recombinant viruses containing
site-directed mutations between 2113 and 285 in the context
of v148(D84-58)-CAT. In v148(D84-58)IICRS-CAT, both A-T-
rich sequences have been deleted and the CRS from the
HCMV major IE promoter has been inserted between 2108
and 295 in an attempt to retain IE86 binding to the promoter.
A second virus, v148(D84-58)CRS-CAT, contains the CRS but
leaves intact the A-T-rich sequences between 294 and 285
(Fig. 1C). In v148(D84-58)IICRS-CAT infections, CAT levels
at all time points tested were comparable to the levels detected
VOL. 72, 1998 HCMV UL112-113 PROMOTER REGULATION DURING INFECTION 2703

FIG. 5. Analysis of CAT protein expression during infection with recombinant viruses containing UL112-113 promoter deletions. Protein lysates were prepared at
8, 24, 48, and 72 h postinfection (hpi), and CAT activity was determined as described in Materials and Methods. Activity is represented as percent acetylation per
microgram of lysate used in the reaction. Solid bars represent the average of two independent infections; error bars represent the range of the two values. Promoter
deletions contained in the recombinant viruses analyzed are indicated on the left.

in cells infected with v148(D84-58)-CAT (Fig. 7). Thus, the
original interpretation was that the mutations had no effect on
late transcription. However, primer extension analysis of RNA
isolated from v148(D84-58)IICRS-CAT infections showed that
transcription from the normal late start site [234 with v148
(D84-58)-CAT] was abolished, and a new RNA start site, with
identical kinetics, was detected upstream at 2140 (Fig. 8). Of
particular interest was the observation that this new start site
was located approximately 39 nucleotides upstream from the
center of the CRS element, similar to the distance between the
normal late start site and the IE86 binding site in v148-CAT. In
addition, a weaker start site with kinetics similar to transcrip-
tion from the 11 early site was detected at 2112. Primer ex-
tension analysis of RNA isolated 72 h after infection with v148
(D84-58)CRS-CAT showed a pattern similar to that of v148
(D84-58)IICRS-CAT, but with low levels of transcripts also ini-
tiating at or near 234.
Thus, replacement of the sequences between 2108 and 295
with the CRS had no effect on the kinetics of late transcription
or on the distance of the late start site from the center of the
element between 2108 and 295. However, when the CRS was
inserted, the late start site was now positioned approximately
39 nucleotides upstream of the element rather than down-
stream. Taken together, these data suggest that the sequences

FIG. 6. Analysis of CAT RNA expression during infection with recombinant viruses containing UL112-113 promoter deletions. Primer extension analysis of total
RNA isolated from cells infected with v148-CAT (A), v119-CAT (B), and v148(D84-58)-CAT (C) was performed as described in the legend to Fig. 4. Promoter deletions
contained in the recombinant viruses analyzed are indicated adjacent to each panel. Arrows indicate the position of the transcriptional start sites detected in the assay.
Products were electrophoresed adjacent to a sequencing ladder (lanes T, G, C, and A) generated with the plasmid p148-CAT and the identical CAT primer to identify
transcriptional start sites. Each panel as well as Fig. 4C was derived from the same autoradiogram but was cropped for display purposes. hpi, hours postinfection.
2704 RODEMS ET AL.
FIG. 7. Analysis of CAT protein expression during infection with v148(D84-58)IICRS-CAT. Protein lysates were prepared at 8, 24, 48, and 72 h after infection with
either v148(D84-58)-CAT or v148(D84-58)IICRS-CAT, and CAT activity was determined as described in Materials and Methods. Activity is represented as percent
acetylation per microgram of lysate used in the reaction. Solid bars represent the average of two independent infections; error bars represent the range of the two values.
Promoter deletions and site-directed mutations contained in the recombinant viruses analyzed are indicated on the left. hpi, hours postinfection.
between 2108 and 295 play a role in directing late transcrip-
tion from the UL112-113 promoter, in a distance-dependent
and possibly orientation-dependent manner.
DISCUSSION
The goal of this study was to more accurately define the roles
of specific sequence elements within the HCMV UL112-113
promoter during infection. The success of the recombinant
virus approach depended on the ability of the UL112-113 pro-
moter inserted into a different region of the genome to func-
tion similarly to the endogenous promoter. We found that
when inserted between the US9 and US10 genes, the UL112-
113 promoter showed identical kinetics of RNA expression as
the endogenous promoter located in the UL region. As with
the endogenous promoter, the inserted UL112-113 promoter
was active both early and late in infection. In particular, a shift
FIG. 8. Analysis of CAT RNA expression during infection with v148(D84-
58)-CAT, v148(D84-58)CRS-CAT, and v148(D84-58)IICRS-CAT. Primer exten-
sion analysis of total RNA isolated from cells infected with v148(D84-58)-CAT
(A), v148(D84-58)IICRS-CAT (B), and v148(D84-58)CRS-CAT (C) was per-
formed as described in the legend to Fig. 4 except that only the 72-h time point
was analyzed for v148(D84-58)CRS-CAT. Promoter deletions contained in the
recombinant viruses analyzed are indicated adjacent to each panel. Arrows
indicate positions of the transcriptional start sites detected in the assay. Numbers
represent the positions of the nucleotides relative to the sequence of the pro-
moter in v148-CAT. Each panel was derived from the same autoradiogram but
was cropped for display purposes. hpi, hours postinfection.
J. VIROL.
in the RNA start site late in infection was also observed from
the inserted UL112-113 promoter. Taken together, our data
indicated that expression of the UL112-113 promoter was ac-
curately represented when located at an alternate position in
the genome and in the opposite orientation relative to the
endogenous promoter. Thus, by inserting the UL112-113 pro-
moter-CAT sequences into the HCMV genome, we were able
to determine which promoter elements were functional at var-
ious times after infection.
UL112-113 promoter activity late in infection. The results
presented here from early time points (8 and 24 h) during
recombinant virus infection are in agreement with data previ-
ously obtained from transient expression analysis (1, 15, 23,
24). Namely, for the UL112-113 promoter, the ATF/CREB site
played a major role early in infection, whereas the sequences
containing the IE86 binding site (2113 to 285) played an
accessory role. However, the most intriguing finding in this
study was that late in infection, the relative importance of the
ATF/CREB site and the sequences containing the IE86 bind-
ing site was reversed. By 72 h after infection, the ATF/CREB
site was no longer functional in transcriptional activation,
whereas the sequences between 2113 to 285 were required
for the switch to the late RNA start site and wild-type levels of
late transcription. This phenomenon was not detected by tran-
sient expression analysis and demonstrates the importance of
using recombinant viruses to accurately determine temporal
regulation of viral promoters during infection.
Two experiments indicated that the ATF/CREB site was no
longer functional late in infection. First, infection with
v148(D84-58)-CAT, which is deleted for the ATF/CREB site,
and v148-CAT, which contains the ATF/CREB site, resulted in
a similar induction of CAT activity by 72 h (Fig. 5). Second,
during infection with a recombinant virus containing only the
ATF/CREB site and TATA box (v119-CAT), CAT levels
steadily declined late in infection despite the increase in viral
template DNA. Furthermore, v119-CAT showed CAT levels at
72 h after infection similar to those of a virus containing only
the TATA box (data not shown). Since CREB is the major
protein that binds to the UL112-113 ATF/CREB site in unin-
fected cells (23), our data suggest that CREB activity is down-
regulated at late times in the infection. Although it has been
known for many years that some early HCMV promoters are
downregulated late in infection (for a review, see reference
20), this study provides the first evidence to suggest that one
mechanism of early promoter shutoff is due to down regulation
of specific host transcription factor activity as the infection
proceeds.
VOL. 72, 1998 HCMV UL112-113 PROMOTER REGULATION DURING INFECTION
FIG. 9. Summary of RNA start sites from recombinant viruses and schematic representation of promoter mutations used in this study. Names of the viruses
containing the promoters are indicated at the left. Major early (E) RNA start sites are denoted by open arrowheads, whereas major late (L) RNA start sites are denoted
by closed arrowheads. Minor RNA start sites are shown as dashed arrows. Sequence elements are as described in the legend to Fig. 1. Numbers represent the positions
of the nucleotides relative to the sequence of the promoter in v148-CAT.
Although CREB binds to the UL112-113 ATF/CREB site in
uninfected cell extracts, it is not known whether CREB still
binds to this promoter late in the infection. The binding activity
of another member of the ATF/CREB family, ATF-1, is in-
duced at late times during infection (11). However, the UL112-
113 ATF/CREB site binds little if any ATF-1 in uninfected
cells (23), and it is not known if ATF-1 can bind to the UL112-
113 ATF/CREB site late in infection. If ATF-1 can bind to the
UL112-113 ATF/CREB site late in infection, our data suggest
that this binding cannot lead to transcriptional activation since
the UL112-113 ATF/CREB site was not functional in late
transcription.
There are several possible mechanisms for the downregula-
tion of CREB activity during infection, including, but not lim-
ited to, degradation, dephosphorylation, or alternative modi-
fication of CREB. CREB activity is normally regulated
through phosphorylation on Ser-133 (6) which is required for
interaction with the CREB binding protein (4). Preliminary
results from our laboratory indicate that CREB protein levels
remain constant throughout infection (19). However, it ap-
pears that Ser-133 becomes dephosphorylated as the infection
proceeds and that there are additional modifications to CREB.
Thus, it is possible that HCMV encodes or modifies functions
to regulate host transcription factor activity so that certain viral
genes are expressed at specific times during the infection. Stud-
ies are in progress to address these questions.
Although the sequences between 2113 and 285, which con-
tain a weak IE86 binding site, are dispensable for early tran-
scriptional activity, results from experiments with two recom-
binant viruses suggested that these sequences are required for
normal levels of late transcription as well as the change in
RNA start site observed at 48 and 72 h after infection. First,
deletion of the sequences between 2113 and 285 in v119-CAT
abolished transcription initiation from the 262 position late in
infection. Second, deletion of the sequences between 284 to
258 in v148(D84-58)-CAT had no effect on the levels of late
transcription, but the late RNA initiation site was moved 28 bp
downstream to 234, a distance similar to the number of de-
leted nucleotides between 284 and 258 (Fig. 6). This putative
2705
distance-dependent late transcription initiation seems to be
unique to the sequences around the weak IE86 site, since no
transcription initiation is detected near the upstream IE86
binding sites (Fig. 4C). However, at present we do not know
whether the three upstream IE86 sites can contribute to tran-
scription initiation either late in infection in the absence of the
sequences containing the weak IE86 binding site or early in
infection in the absence of the ATF/CREB site.
Since the sequences from 2113 to 285 are protected by
IE86 in a DNase I footprint, it is possible that IE86 binding to
this sequence plays a role in late transcription initiation from
the UL112-113 promoter. Alternatively, there are two A-T-
rich sequences within that region which could behave as
TATA-like sequences to direct late transcription in a distance
dependent manner. To distinguish between these possibilities,
we constructed two recombinant viruses that contained substi-
tution mutations between 2113 and 285, v148(D84-58)CRS-
CAT and v148(D84-58)IICRS-CAT. Although the sequences
from 2113 to 285 are protected by IE86 in a DNase I foot-
print, this sequence diverges somewhat from the consensus
IE86 binding site. Based on footprint analyses, a consensus
IE86 binding site consists of an A-T-rich 10-nucleotide se-
quence bounded by CG dinucleotides (1, 16, 24). The sequence
between 2113 and 286 contains a CG with A-T rich sequences
on either side but lacks a complementary CG dinucleotide
(Fig. 1). The best approximation of the IE86 binding site within
the 2113 to 284 sequence, based on the DNase I protection
pattern (24), would be nucleotides 2108 to 295. Therefore, we
replaced that sequence with the CRS in v148(D84-58)CRS-
CAT to determine if the IE86 binding site was involved in late
transcription. We also constructed another virus, v148(D84-
58)IICRS-CAT, which included mutation of the TATA-like
sequences from 288 to 284 in addition to the substituted
CRS.
The results of the primer extension analyses on all recom-
binant viruses are summarized in Fig. 9. Substitution of the
sequences between 2108 and 285 resulted in several interest-
ing observations. First, transcription from the late RNA start
site at 234 was abolished in v148(D84-58)IICRS-CAT, sug-
2706 RODEMS ET AL.
gesting that the sequences from 2108 to 285 were involved in
directing late transcription initiation at the downstream site.
We also noted that there was a very low level of steady-state
RNA initiating near 234 in v148(D84-58)CRS-CAT infection.
One interpretation of these data is that the TATA-like se-
quence between 288 and 284 may play a role in determining
the site of late transcription initiation and that the upstream
sequences between 2108 to 295 are important for high-level
expression. However, because close inspection of the gels sug-
gests that this RNA may actually be initiating at 233, it is
possible that the substituted sequences have activated a weak
cryptic site for initiation at late times. Nevertheless, it appears
that the wild-type sequences containing the weak IE86 binding
site between 2108 and 295 are involved in downstream late
transcription initiation.
One of the most interesting observations from the primer
extension analysis of v148(D84-58)CRS-CAT and v148(D84-
58)IICRS-CAT infections was the emergence of two new RNA
start sites upstream of the inserted CRS. A weak start site with
early kinetics was detected initiating from 2112. However,
since this site is observed only in v148(D84-58)IICRS-CAT, it
is likely the result of mutation of the sequences between 294
and 285. The strongest of the two new sites initiated at 2140
and displayed late kinetics. Thus, late transcription was abol-
ished from a position downstream of the IE86 binding site and
instead initiated at a novel position upstream. Interestingly, the
novel late RNA start site initiated within the polylinker se-
quence which had been inserted as a result of cloning the
UL112-113 promoter into the transfer vector used for recom-
binant virus construction. Since this novel late RNA start site
was detected during infection with both v148(D84-58)CRS-
CAT and v148(D84-58)IICRS-CAT, insertion of the CRS was
responsible for the appearance of this site. It is interesting that
the distance from the novel late start site (2140) to the center
of the inserted CRS (between nucleotides and 2102 and
2101) is almost identical to the distance from the center of the
putative weak IE86 binding site (between nucleotides and
2102 and 2101) to the wild-type late start site (262) detected
in v148-CAT infection. This observation raises the possibility
that the orientation of the CRS, and of the weak IE86 binding
site within the wild-type UL112-113 promoter, determines the
positioning of the late transcriptional start site. Since IE86
binds to the minor groove of the DNA (17), the orientation of
the IE86 binding site relative to other surrounding sequences
may result in certain structural effects on IE86 binding that
determine the position of late transcription initiation. Alterna-
tively, unidentified sequences within the CRS, and within the
UL112-113 promoter sequences between 2108 and 295, may
be involved in orientation-dependent, late transcription initia-
tion independent of IE86 binding. At present, we also cannot
rule out the possibility that the mutations cause activation of
an upstream cryptic promoter which results in initiation of
transcription at position 2140. Further experiments are under
way to determine the precise sequences required, the depen-
dence on orientation and distance, and whether IE86 binding
to these sequences is essential.
UL112-113 promoter activity early in infection. Analysis of
CAT activity and mRNA levels at 8 and 24 h after infection
with v148(D84-58)-CAT, which is deleted for the ATF/CREB
site, suggested that the ATF/CREB site is required for high
levels of promoter activity early in infection, whereas the se-
quences containing the IE86 binding site are less important.
Since deletion of the ATF/CREB site positioned the IE86
binding site closer to the TATA box, one can argue that the
IE86 site is no longer functional due to the change in its
position. However, in transient expression assays, site-directed
J. VIROL.
mutation of the ATF/CREB site without altering the position
of the IE86 binding site showed that the IE86 site could not
compensate for the loss of the ATF/CREB site (23). Since our
results suggest that UL112-113 promoter activity in transient
expression assays mimics that of early time points during in-
fection, we expect that the IE86 site in its normal location also
would not compensate for the loss of the ATF/CREB site early
in infection.
Since the ATF/CREB site is functional only early during
infection and since the host transcription factor CREB binds to
this site within the UL112-113 promoter, HCMV, like many
viruses, employs the strategy of utilizing cellular transcription
factors in the initial phases of the infection. However, only
weak expression from the UL112-113 promoter is observed
when viral protein synthesis is inhibited (27), suggesting that
other viral proteins, such as IE86, are required for maximum
levels of transcription. Our results also suggested that the se-
quences containing the weak IE86 binding site played only a
modest role early in infection. Similarly, in transient expression
assays, deletion of the weak IE86 binding site reduced UL112-
113 promoter activity only about twofold (23). However, in the
transient expression experiments the IE86 protein is required
for promoter activity even in the absence of an IE86 binding
site on the promoter. Thus, IE86 may function through pro-
tein-protein contacts without the requirement for DNA bind-
ing, or at least without the need for a consensus IE86 binding
site. Indeed, IE86 can interact with the CREB binding protein
(CBP) and weakly with a truncated form of CREB, DCREB
(15, 23). Since IE86 is expressed in the recombinant virus
infections, these studies do not allow us to distinguish the role
of the IE86 protein at the promoter early in infection. We can
only conclude that the sequences containing the weak IE86
binding site are not essential for early UL112-113 promoter
activity.
The results presented here suggest that for specific promot-
ers there are different mechanisms governing early and late
viral transcription. It is likely that many early promoters use
existing cellular transcription factors, as well as virus-encoded
factors, to enhance transcription under low-template condi-
tions prior to viral DNA replication. However, as the infection
proceeds, certain cellular factors may be either downregulated
or upregulated to alter the expression pattern of specific viral
genes. Although there is an increase in viral DNA template
late in infection due to DNA replication, transcription from
the 11 position in the UL112-113 promoter decreases. The
mechanism governing this downregulation of transcription
from one site and upregulation from a different site late in
infection is unclear. Further analysis of other early and late
HCMV promoters is needed to fully understand the regulation
of viral gene expression during infection.
ACKNOWLEDGMENTS
We thank Ruth Schwartz and Ed Mocarski for plasmids used in this
study. We also thank Roopashree Dwarakanath, Elizabeth Fortunato,
Anita McElroy, Chris Morello, and Bryan Salvant for helpful discus-
sions and critical reviews of the manuscript.
This investigation was supported by NIH grant CA 34729 (D.H.S.)
and NIH training grant AI-07384 (S.M.R.).
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