JOURNAL OF VIROLOGY, Mar. 2007, p. 2592–2604
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 6
High-Frequency Epigenetic Repression and Silencing of Retroviruses
Can Be Antagonized by Histone Deacetylase Inhibitors and
Transcriptional Activators, but Uniform Reactivation in
Cell Clones Is Restricted by Additional Mechanisms?
Richard A. Katz,1* Emily Jack-Scott,1† Anna Narezkina,1§ Ivan Palagin,1§ Pamela Boimel,1¶
Joseph Kulkosky,2Emmanuelle Nicolas,1James G. Greger,1‡ and Anna Marie Skalka1
Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111,1
and Chestnut Hill College, Philadelphia, Pennsylvania 191182
Received 1 August 2006/Accepted 13 December 2006
Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that
influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and
high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector
DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus
(hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene,
and cells were sorted into two populations based on GFP expression [GFP(?) and GFP(?)]. Two potent
epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(?)
population as well as individual GFP(?) clones and (ii) high-frequency GFP reporter gene silencing in
GFP(?) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon
after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding,
we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP
activation in GFP(?) cells and can also increase GFP expression in GFP(?) cells. In the case of the GFP(?)
populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used
that finding to enrich for stable GFP(?) cell populations in which viral GFP reporter expression could be
reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the
promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal
repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin.
Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating
that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into
S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain
promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by
After integration, retroviral DNA becomes a segment of the
host chromosome and is therefore duplicated during S phase
and passed to daughter cells following mitosis (12). In addition
to establishing a permanent association between viral and host
DNA, integration allows for efficient retroviral gene expres-
sion. However, DNA integration does not ensure continued
viral gene expression. Gene silencing is frequently observed
when retroviruses are used as vectors for gene delivery; trans-
duction of the introduced reporter gene is successful, but ex-
pression is extinguished at various times postinfection (19, 49,
57, 59). This phenomenon is prominent in embryonic or adult
stem cells and has been most thoroughly studied in this context
(9, 19, 24, 49, 50, 58, 63). Silencing is typically mediated by
DNA methylation or chromatin modifications at the viral loci
(19, 49, 57, 59). As this repressed viral state is heritable over
many cell generations, retroviral silencing is by definition epi-
genetically controlled and may signify an active cellular mech-
anism to repress foreign DNA (27, 65). Epigenetic silencing is
generally reversible, and retroviral gene expression can be re-
activated by various stimuli.
Although retroviral DNA silencing has been well studied,
the parameters that influence the initiation, maintenance, and
reactivation are not fully understood. The earliest attempts to
introduce murine leukemia virus into developing mouse em-
bryos and stem cells (28) led to the discovery of a correlation
between retroviral silencing and DNA methylation (59). Mu-
rine leukemia virus cis-acting sequences that mediate the
repressive effects were found to reside within the viral long
terminal repeat (LTR) promoter and other regions (49). How-
ever, more recent studies have implicated histone modifica-
tions in retroviral silencing (19, 22, 23, 49, 50, 59, 61, 64). A
family of transcriptional activators are enzymes, histone acetyl-
transferases (HATs), that acetylate histone tails (29). This
acetylation is thought to promote chromatin opening, as well
* Corresponding author. Mailing address: 333 Cottman Avenue,
Philadelphia, PA 19111. Phone: (215) 728-3668. Fax: (215) 728-2778.
† Present address: Yale University, New Haven, CT.
§ Present address: Smolensk State Medical Academy, Smolensk,
¶ Present address: Albert Einstein College of Medicine, New
‡ Present address: Wyeth Research, Collegeville, PA.
?Published ahead of print on 3 January 2007.
as to serve as a biochemical mark for recruitment of other
transcriptional activators. Histone deacetylases (HDACs) re-
move these acetyl groups and function as transcriptional re-
pressors that maintain epigenetic silencing. Thus, HATs and
HDACs can act as local antagonists to regulate gene expres-
sion. Treatment with histone deacetylase inhibitors (HDIs)
results in broad histone acetylation and concomitant activation
of a subset of silent cellular genes, as well as silent reporter
genes (7, 8, 66). Although DNA methylation can recruit
HDAC repressor complexes, there are examples of DNA
methylation-independent HDAC-mediated reporter gene re-
pression (50). Numerous studies have now indicated both in-
dependent and combinatorial roles for DNA methylation, his-
tone modifications, and chromatin remodeling in retroviral
silencing (9, 19, 24, 25, 40–42, 45, 49, 50, 57–59, 63). However,
a universal model for the initiation and maintenance of retro-
viral silencing has not yet emerged. The apparent diversity of
mechanisms may be due to differing cell- and virus-specific
features. In addition, redundancies in host silencing mecha-
nisms or temporal-specific effects may cause difficulties in cor-
relating the various findings.
Two ways in which epigenetic modifiers (e.g., HATs,
HDACs, chromatin-remodeling complexes, and DNA methyl-
transferases) might associate with retroviral DNA to initiate
and maintain epigenetic silencing are by “spreading” from
regions of host chromatin that flank the integration site (17)
and/or by more direct recruitment to viral DNA (22, 49). Tran-
scriptional activity of neighboring genes might also influence
expression of the integrated viral DNA (37). Therefore, fea-
tures that influence the frequency at which retroviral silencing
occurs may include the location of the retroviral DNA integra-
tion site within the host chromosome (18, 30, 31, 37), the
orientation of the integrated viral DNA with respect to host
DNA (20), and the propensity of the viral LTR or other cis-
acting sequences to recruit repressive host factors (49). The
so-called “position effects” on retroviral expression are be-
lieved to be mediated by the local chromatin state; i.e., consti-
tutive/facultative heterochromatin (closed) or euchromatin
(open), corresponding to silent and potentially active regions,
respectively. Features of constitutive heterochromatin include
DNA methylation, presence of repressive histone code modi-
fications, and late S-phase replication.
Initial studies revealed that the DNA of human immunode-
ficiency virus type 1 (HIV-1) integrates preferentially into ac-
tive transcription units (protein-coding genes) (56), although
the biochemical basis for this preference is not yet understood.
We (47) and others (4, 44) investigated avian sarcoma virus
(ASV) integration site selection in human cells, for which a
modest preference for integration into genes was observed.
However, transcriptional activity of these target genes at the
time of integration neither stimulated nor inhibited ASV inte-
gration (47). Postintegration latency of HIV-1 is controlled in
part by epigenetic mechanisms, including histone modifica-
tions (14, 23, 36, 51, 61, 64), and some studies have suggested
that integration near closed chromatin may account for estab-
lishment of such rare HIV-1 latency (30, 31). Another study
indicates that a wide variety of flanking genomic features can
influence HIV expression (37).
While using human HeLa cells to study retroviral DNA
nuclear import (32) and integration site selection (47), we
found that an ASV-based green fluorescent protein (GFP)
reporter vector was susceptible to rapid silencing and variega-
tion. Other studies from our laboratory demonstrated that
HDACs can physically associate with ASV DNA complexes
early after infection (22). Here we report results from a series
of cell population and clonal studies, which show that the
epigenetically silent state of GFP reporter genes can be antag-
onized by HDIs, resulting in robust activation of GFP expres-
sion. We observed similar reactivation after treatment with the
phorbol ester prostratin, although in this case, the response
was promoter specific. By following cells microscopically dur-
ing reactivation, we determined that the response to these
compounds is not uniform in cell clones, and this may indicate
an orderly switching between transcriptionally responsive and
nonresponsive states. These studies provide further insights
into the mechanisms of retroviral silencing as well as an expla-
nation for the nonuniform response to HDIs.
MATERIALS AND METHODS
Viruses and cells. The ASV-based vectors that utilize the LTR (ASVA-GFP)
or an internal human cytomegalovirus (hCMV) immediate-early (IE) promoter
(ASVA-CMVEGFP) to drive expression of the enhanced GFP gene have been
described previously (33). These vectors are derivatives of an ASV-based vector
that is capable of only one round infection of mammalian cells (1). A third
ASV-based vector was constructed for this study, in which the hCMV IE pro-
moter in ASVA-CMVEGFP was replaced with the elongation factor 1 (EF-1)
alpha promoter by using standard cloning methods. The EF-1 alpha promoter
region was PCR amplified from the pEF1 plasmid (Invitrogen). The HIV-1-
based GFP vector was described previously (32). Jurkat T-cell clones containing
a latent HIV-1 genome encoding a GFP reporter were kindly provided by Eric
Chemicals and antibodies. Trichostatin A (TSA), aphidicolin, nocodozole,
valproic acid, sodium butyrate, and apidicin were purchased from Sigma-Aldrich.
Tumor necrosis factor alpha (TNF-?), phorbol 12-myristate 13-acetate (PMA),
and prostratin (12-deoxyphorbol 13-acetate) were purchased from Biomol, and
the anti-GFP antibody was purchased from Abcam.
Fluorescence-activated cell sorter (FACS) analysis. GFP expression was mea-
sured using a Becton Dickinson FACScan flow analyzer. Data were analyzed
using FlowJo software.
Infections, cell sorting, and cell cloning. HeLa cells were infected with GFP
reporter vectors by incubation with several dilutions of virus stocks in the pres-
ence of 10 ?g/ml DEAE-dextran. The ASV-based vectors were produced in
DF-1 chicken cells, and the HIV-1-based vectors were produced using the three-
plasmid transfection strategy, as described previously (32). After 48 h, cultures
were examined for GFP expression by microscopy, and the percentage and
intensity of GFP expression was quantitated by FACS analysis. The GFP expres-
sion patterns shown in Fig. 1A were observed reproducibly. Cultures that con-
tained 10 to 30% GFP(?) cells were selected for further analysis. Viable cells
were preparatively sorted at 7 to 10 days postinfection using a Becton Dickinson
FACS-VantageSE flow cytometer. Several independent infections and sorting
experiments were used to prepare GFP(?) and GFP(?) populations. The gen-
eral gating strategy is outlined in Fig. 1B. For the GFP(?) fraction, cells were
selected from approximately the two most intense decades of GFP signal, as
these cells expressed sufficient GFP to be analyzed by microscopy. The GFP(?)
cells were either sorted as a population or distributed for cloning as individual
cells in 96-well dishes.
A multistep sorting strategy was used to purify cells in which GFP expression
could be reactivated by TSA treatment. The GFP(?) cell population was treated
with TSA, and the GFP-expressing cells were sorted. This population was pas-
saged for ca. 10 days, at which time there was a significant loss of GFP expres-
sion. The residual GFP-expressing cells were then removed by cell sorting, and
the resulting population was designated TI, as they were selected for TSA-
induced GFP expression. On occasion, a second sorting step was required to
eliminate contaminating GFP-expressing cells. An identical strategy was used to
isolate TI-C, TI-L, and TI-E cells. We noted that during initial isolation of
GFP(?) cells, gating of the entire symmetrical GFP(?) peak corresponding to
uninfected HeLa cells (Fig. 1B) reproducibly generated cell populations in which
a subset of cells responded to TSA. Higher-stringency gating to exclude cells with
VOL. 81, 2007 VARIEGATED REACTIVATION OF SILENT RETROVIRUSES2593
greater fluorescence within the GFP(?) peak appeared to reduce the fraction of
cells that responded to TSA, indicating that TSA-responsive cells were not
uniformly distributed within the GFP(?) peak.
Cell synchronization and microscopy. Synchronization of HeLa cells was car-
ried out essentially as described previously (32). Subconfluent HeLa cultures
were washed with medium three times to remove weakly adherent cells and were
then treated with nocodozole (16 ng/ml) for 2 h. Mitotic shakeoff was carried out,
and the resultant cells were plated under dilute conditions to produce well-
isolated microclones. Microscopy was carried out using a Nikon Eclipse TE800
inverted microscope fitted with a Nikon COOLPIX 950 digital camera.
qPCR. The 5?-nuclease assays with TaqMan chemistry were used to measure
the relative copy number of GFP compared to a control HeLa gene (human
albumin, 4q11-q13) (39). DNA was prepared from GFP(?), GFP(?), or un-
sorted cells by using a DNeasy kit (QIAGEN). The Primer Express software
(Applied Biosystems) was used to design primers and probes. The quantitative
real-time PCR (qPCR) primer and probe sequences for albumin were 5?-CAT
TTATTGGTGTGTCCCCTTG-3? (forward), 5?-ACACCAGTGAAAACAATT
TAAGCC-3? (reverse), and (6-FAM)CCCAACAGAAGAATTCAGCAGCCG
TAAG(BHQ1) (probe), and those for enhanced GFP were 5?-CCCAGTCCGC
CCTGAG-3? (forward), 5?-ACGAACTCCAGCAGGACCA-3? (reverse), and
(6-FAM)CCCCAACGAGAAGCGCGATCA(BHQ1) (probe). The 5? and 3?
ends of the probes were labeled with the reporter dye 6-FAM (6-carboxyfluo-
rescein) (Glenn Research) and the quencher dye BHQ1 (Black Hole Quencher)
(Biosearch Technologies), respectively. All primers and probes were synthesized
by the Fox Chase Cancer Center Fannie Rippel Biotechnology Facility. Quan-
titect (QIAGEN) master mix was used for PCR. The Cepheid Smart Cycler was
used for qPCR analyses. Cycling conditions were 95°C for 10 min, followed by 45
(two-step) cycles (95°C for 20 s and 62°C for 60 s). The difference in threshold
cycle (CT) values (?CT) between GFP and albumin was used to normalize the
amount of genomic DNA. A GFP standard was established by infecting HeLa
cells at a very low multiplicity of infection (MOI) with the HIV-GFP vector and
sorting the GFP(?) cell population (1% of the total), ensuring that a single copy
of GFP was present. The ratio of GFP to genomic DNA (albumin) in this sample
is normalized to 1 (one copy of GFP per cell genome), and this sample is taken
as the calibrator. The differences in ?CTs (??CT) for the samples of interest and
the calibrator are used to estimate the relative quantity (RQ) of GFP (to the
calibrator sample) by using the formula RQ ? 2???CT(39). The values and
standard deviations in Table 1 (copy number) were obtained by averaging results
from three PCRs (for each reporter) performed with inputs of 100, 20, and 4 ng
of genomic DNA (based on optical density). As a negative control, DNA was
prepared, using sorted GFP(?) cells from a culture that was infected with an
HIV-GFP vector [50% GFP(?) cells]. As we could find no evidence for silent
GFP in this GFP(?) population by conventional PCR or TSA treatment, this
sample served as a rigorous negative control. Analysis of this control sample gave
a CTvalue comparable to that of the GFP standard sample for albumin, but the
CTvalue for GFP was lower by ca. 7 cycles (??CT? 7), indicating that the
relative GFP copy number was negligible (27-fold lower).
Broad range of GFP reporter expression intensities in HeLa
cells after infection with an ASV-based vector. Interspecies
infection with retroviruses can be accomplished through
pseudotyping, and such a strategy has been used for a variety
of laboratory and gene transfer experiments. During our pre-
vious studies (32, 47), we frequently observed clonal variega-
tion (mosaic patterns) of GFP reporter expression (by micros-
copy) early after infection of human HeLa cells with a
pseudotyped ASV-based vector (1). Such variegated expres-
sion patterns were somewhat unexpected, as the GFP gene was
driven by the strong hCMV IE promoter. As infection is lim-
ited to one round in this system, reinfection cannot contribute
to this phenomenon. In addition, infection of HeLa cells with
an ASV-GFP vector that is defective for DNA integration does
not result in detectable GFP expression (33), and therefore the
observed pattern could not be attributed to dilution of unin-
tegrated viral DNA during cell outgrowth. We suspected that
the rapid variegation was produced by epigenetic changes and
might represent intermediate steps in GFP reporter gene si-
To examine this phenomenon in more detail, we sought to
establish infection conditions whereby various GFP expression
patterns could be more readily interpreted. In particular, it was
important to limit the MOI, such that differences in GFP
expression intensity could not be due to differences in GFP
copy number. Accordingly, HeLa cells were infected with var-
ious amounts of the ASV-GFP vector to derive conditions
whereby only ca. 10 to 30% of the cells expressed GFP at 7 to
10 days postinfection. In these populations we designate the
resulting GFP-expressing cells GFP(?) and nonexpressing
cells GFP(?). The latter cell population would be expected to
include uninfected cells, as well as cells in which the viral DNA
may be silenced by epigenetic processes.
FIG. 1. GFP intensity profiles of infected HeLa cells as analyzed by
FACS. (A) Profiles of GFP expression were measured at 48 h postin-
fection. Shown are GFP profiles for uninfected HeLa cells (CON)
(light shading) and for cells after infection with increasing amounts of
the ASV-GFP vector. A parallel culture was infected with an HIV-1-
based vector (HIV-GFP) (dark shading). Data were analyzed using
FlowJo software. y-axis scaling was used to more readily compare the
intensity profiles. (B) Gating strategy to isolate GFP(?) and GFP(?)
cells. A representative profile from panel A is shown with the approx-
imate gates used for isolation of cell populations by preparative cell
sorting. In this example, the GFP(?) cells represented ca. 30% of the
culture. A subfraction of brighter GFP(?) cells were selected for
isolation to ensure that GFP patterns were bright enough to observe
2594 KATZ ET AL.J. VIROL.
After infection with the ASV-GFP vector, FACS analysis
showed that the GFP intensity profile was quite broad at early
times, and this pattern was generally independent of the MOI.
(Fig. 1A). Such broad profiles are typically attributed to dif-
ferences in reporter gene expression that result from integra-
tion at different sites within the host cell genome and/or posi-
tional variegation (mosaic patterns of reporter expression
produced during outgrowth of cell clones within the popula-
tion). Strikingly, introduction of an HIV-based vector that en-
coded a similar hCMV IE-driven GFP reporter gene (32)
produced a more intense and uniform peak of GFP-expressing
cells (Fig. 1A); after several additional days of culture, these
intense cells became the predominant form (not shown) (16).
The resulting characteristic GFP intensity profiles produced by
these ASV- and HIV-based vectors remained fairly constant
during long-term passage of these cell populations (not
shown). The distinct GFP profiles observed with the ASV- and
HIV-based vectors suggest that the epigenetic effects are not
simply a response to the heterologous GFP gene (15).
Evidence for epigenetic effects on ASV-based vector GFP
reporter gene expression. We hypothesized that the broad
range of GFP intensities observed in the population of ASV-
based vector-infected cells was due to either (i) outgrowth of
cell clones with characteristic GFP expression levels, (ii) posi-
tional variegation of GFP expression within cell clones, (iii)
cell cycle effects on GFP expression, (iv) cells which represent
intermediates in GFP silencing, or (v) a combination of these
features. Furthermore, because of the broad expression range,
we suspected that a fraction of the GFP(?) cells contained
silent GFP reporter genes. To test these hypotheses, infected
cultures containing ca. 30% GFP(?) cells were preparatively
sorted at 2 weeks postinfection, and single GFP(?)-positive
cells were deposited robotically in each well of a 96-well dish
(Fig. 1B). Inspection of the wells immediately and at 1 day
postplating revealed that ca. 30 wells received single, viable
cells. Eleven single-cell-derived GFP(?) cell clones were ulti-
mately expanded from these wells and were designated AP1
through AP11. By microscopy, five of the clones (including
AP6, AP10, and AP11) contained apparent mixtures of bright
and dim GFP(?) cells (designated variegated [V] clones),
while six clones (including AP2 and AP4) displayed more in-
tense and uniform GFP expression (designated uniform [U]
We devised a strategy to follow development of the varie-
gated patterns during outgrowth of single cells into colonies.
Cultures from the GFP(?) cell pool were treated briefly with
nocodazole to enrich for mitotic cells, and a mitotic shakeoff
was performed. Mitotic cells were plated under dilute con-
FIG. 2. Variegation of GFP expression during outgrowth of GFP(?) microclones. (A) A mitotic shakeoff strategy was used to promote highly
synchronized outgrowth of microclones from single cells. Shown is a single field, imaged ca. 2 h after plating mitotic cells. Typically, synchronization
occurred with 70% to 90% efficiency. Boxed colonies have completed cytokinesis synchronously and entered G1as indicated by the cell doublets.
(B) GFP patterns were monitored during colony outgrowth. Left panels show eight-cell colonies, with the upper panel illustrating a colony
displaying uniform GFP expression and the middle and bottom panels highlighting variegated colonies. Right panels show a representative
variegated pattern in a large colony. Phase-contrast and fluorescent images are shown. (C) Analysis of GFP variegation during outgrowth of
synchronous microclones derived from GFP(?) cell clones. One uniform (U) (AP2) and one variegating (V) (AP6) clone were examined.
Representative images are shown. Arrows indicate cells displaying higher-intensity GFP expression.
VOL. 81, 2007VARIEGATED REACTIVATION OF SILENT RETROVIRUSES 2595
ditions, and they entered G1with 70 to 90% synchrony, pro-
ducing cell doublets (Fig. 2A). This method ensures that the
majority of adjacent cells are daughters that will grow synchro-
nously over several cell divisions; the synchronous growth also
minimizes potential cell cycle effects on GFP intensity. Micro-
clones that were derived from the GFP(?) population were
analyzed at the four- and eight-cell stages. As shown in Fig. 2B,
we observed two predominant GFP intensity patterns during
outgrowth of these clones: uniform GFP cell-to-cell intensity
within each colony, or variegated GFP intensities (both shown
at the eight-cell stage). The variegated pattern was typically
characterized by four bright cells and four dim cells (Fig. 2B,
left panels), and the sectored pattern implies that the bright
and dim cells were derived from single progenitors at the
two-cell stage, respectively. When such colonies became larger,
GFP expression was detected throughout the colony, and a
classical variegated pattern was prominent (Fig. 2B, right pan-
els). Typically, sectoring of adjacent cells of similar intensity
could be observed. We interpret these results to indicate that
these variegated patterns in cell clones were due to orderly
epigenetic-based oscillations of GFP expression (positional
To investigate this phenomenon further, we performed sim-
ilar experiments with two GFP(?) cell clones, AP2 (U) and
AP6 (V) (Fig. 2C). Here, we examined two-cell colonies for
evidence of oscillation. In the case of the AP6 V clone, we
observed frequent cell doublets in which one cell was very
bright and one cell was dim; in contrast, the U clone typically
produced cell doublets with similar GFP intensities. In the V
clone, the substantial differences in GFP expression between
the two daughter cells could more readily be accounted for by
derepression of GFP in the bright daughter cell, as opposed to
silencing, GFP dilution, and turnover in the dim cell. We
conclude, therefore, that a fraction of HeLa cells contain in-
tegrated viral genomes that are prone to epigenetic variega-
tion, and one possible interpretation is that such variegation is
the result of oscillation between repressed and derepressed
Evidence for high-frequency epigenetic silencing of the ASV
vector in HeLa cells. We next asked if the epigenetic effects on
GFP reporter gene expression could include complete silenc-
ing. If so, it would be expected that in addition to uninfected
cells, the GFP(?) population would include cells that contain
integrated viral DNA. Cellular DNA was prepared from
GFP(?) and GFP(?) populations in which the amount of
infecting virus had been adjusted to produce ca. 20 to 35%
GFP-positive cells, to minimized the fraction of cells that
harbor multiple copies of integrated DNA (see Fig. 1B for a
representative FACS profile). GFP DNA could be readily
detected in GFP(?) cells by semiquantitative PCR (not
shown), indicating that these cells contained epigenetically
silent viral DNA.
The average copy numbers of GFP DNA in the GFP(?) and
GFP(?) populations were next measured by qPCR (Table 1).
To facilitate quantitation, a single-copy GFP reporter gene
standard was calibrated to the cellular albumin gene. As ex-
pected, qPCR analysis using two independent GFP(?) cell
populations indicated that these sorted cells contained ca. 1
copy of GFP DNA on average. Analysis of the GFP(?) cells
from a starting population containing 35% GFP(?) cells re-
vealed an average GFP copy number of ca. 0.3, confirming that
a significant fraction of cells contained silent viral genomes.
From these experiments, we can estimate the fraction of
ASV integration events that result in silencing of GFP. As the
initial conditions for infection produced approximately 35%
GFP(?) cells, a copy number of 0.3 in the GFP(?) population
indicates that the distribution in the starting culture was ca.
35% GFP(?), 20% GFP silent, and 45% uninfected.
Induction of GFP expression by trichostatin A. Variegation
of GFP expression in the ASV vector-infected population could
silent cells could be identified by 7 to 10 days postinfection. These
rapid epigenetic changes were consistent with our previous find-
ings that HDACs, key regulators of epigenetic silencing, can as-
sociate with ASV DNA soon after infection of HeLa cells (22).
tails, thereby promoting transcriptional repression. Treatment of
cells with HDIs results in increased global histone acetylation and
are normally repressed by HDACs.
To determine if HDACs have a role in maintaining silencing
of the GFP reporter genes, we treated GFP(?) cell popula-
tions with a known HDI. For these experiments, GFP(?) cells
were sorted from infected cultures that contained 20 to 30%
GFP(?) cells. The sorted GFP(?) cells were then treated with
TSA, an HDI frequently reported to activate epigenetically
silenced host cell and reporter genes. TSA treatment resulted
in a significant increase in total histone H4 acetylation within 3
to 4 h (data not shown). We observed that TSA treatment
induced GFP expression in the GFP(?) population, in a dose-
dependent manner as determined by FACS analysis (Fig. 3A).
Western blot analysis (not shown) verified that GFP protein
was initially undetectable in these GFP(?) cells but accumu-
lated after TSA treatment. Quantitation by FACS analysis
indicated that GFP expression was induced in ca. 10% of the
GFP(?) cells. This percentage was lower than that found to
contain the GFP gene by qPCR (ca. 30%). We considered that
either some ASV genomes are silenced by a mechanism that is
nonresponsive to TSA treatment, the treatment was not opti-
TABLE 1. Copy numbers of integrated ASV-GFP reporter genes in
GFP(?) and GFP(?) cellsa
Fraction of GFP-positive
cells in unsorted
GFP copy no. per cell
(mean ? SD)
1.4 ? 0.2
0.3 ? 0.0
1.2 ? 0.3
aThe GFP reporter gene copy number relative to the cellular albumin gene
was determined by qPCR (see Materials and Methods). As a negative control,
GFP(?) cells were sorted from a culture infected with an HIV-GFP vector in
which ca. 50% of cells were GFP(?). Results indicated that GFP(?) cells
derived from this culture did not harbor significant levels of HIV DNA (27-fold
lower than the GFP standard sample).
bTo prepare control cells containing a single-copy GFP gene, HeLa cells were
infected with an HIV-GFP vector under conditions where ca. 1% of the cells
were GFP(?). The GFP(?) population was isolated by cell sorting and used to
prepare the DNA standard.
cThe indicated GFP(?) and GFP(?) cell populations were sorted from the
same starting culture.
dNR, not relevant.
2596KATZ ET AL.J. VIROL.
mal, or the response was limited by other cellular processes
To determine if TSA-induced GFP expression was heritable
over many cell divisions, the drug was removed after 24 h and
cultures were propagated and observed. Significant loss of
GFP expression was observed over a period of ca. 1 week (not
shown). As described below in detail, we determined by cell
sorting that this loss was not due to inviability of cells in which
GFP expression was induced. We conclude, therefore, that
although treatment with the HDI was sufficient to activate
expression, a remaining epigenetic signature could mediate
Enrichment for cell populations in which retroviral silenc-
ing is controlled by HDACs. The finding that withdrawal of
TSA resulted in resilencing of GFP expression in a subset of
cells allowed us to enrich for cells in which the ASV-GFP
reporter gene silencing is regulated by HDACs, as follows. A
GFP(?) cell population was sorted from a starting culture
which contained ca. 20% GFP(?) cells. This GFP(?) popula-
tion was treated with TSA, and the resulting subpopulation of
GFP-expressing cells was isolated by cell sorting. Continued
culturing of this population over ca. 10 days in the absence of
TSA resulted in the progressive loss of GFP expression in a
large percentage of the cells, as expected. Residual GFP-ex-
pressing cells were then removed by sorting. The resulting
GFP(?) subpopulation was then passaged for various times
and rechallenged with TSA. As shown in Fig. 3B, we observed
significant TSA-inducible GFP reactivation even after long-
term passage of these cells. The extent of the response never
approached 100% (see below) and varied from experiment to
experiment (ca. 30% to 60% [data not shown]). We have des-
ignated this enriched population TI-C cells (for TSA-inducible,
hCMV IE-driven GFP). Using the method described in Table
1, the average GFP reporter gene copy number in the TI-C
population was determined to be 0.8 ? 0.2. As shown in Fig.
3B, treatment of these cells with chemically diverse HDIs (so-
dium butyrate, apidicin, and valproic acid) also induced GFP
To confirm that GFP protein expression was mediated by a
GFP mRNA that initiated in the integrated viral vector DNA,
Northern blot analysis was performed with total cell RNA
isolated from untreated and TSA-treated TI-C cell popula-
tions. As shown in Fig. 3C, TSA treatment resulted in the
appearance of an RNA transcript of the size expected if tran-
scriptional initiation occurred at the internal hCMV IE pro-
moter, and 3?-end processing was directed by the viral 3? LTR.
This analysis revealed that repression and reactivation by TSA
were tightly regulated at the level of transcription. We note
that an LTR-driven RNA transcript corresponding to full-
length vector RNA was not readily detectable. This was not
unexpected, as we have found independently using an LTR-
driven GFP vector, that full-length viral RNA is difficult to
detect in this system, possibly due to the weaker LTR promoter
and excessive splicing of ASV RNA in mammalian cells (3).
Evidence for HDAC-mediated repression in GFP(?) cells.
As illustrated in Fig. 1 and 2, a broad range of GFP intensities
as well as positional variegation of GFP expression was ob-
served in the GFP(?) population of HeLa cells infected with
the ASV-GFP vector. To determine if these effects could also
be modulated by cellular HDACs, GFP(?) cell populations
and clones were treated with TSA. A dramatic increase in GFP
intensity in the GFP(?) cell population was observed following
such treatment (data not shown). Similarly, treatment of rep-
resentative GFP(?) V cell clones, in which GFP intensity was
FIG. 3. Reactivation of GFP reporter genes after treatment with
HDIs. (A) Cells were sorted at 8 days postinfection, and the GFP(?)
cells were passaged for several months. GFP(?) cells were treated with
the indicated concentrations of TSA, and GFP expression was quan-
titated by FACS at 24 h posttreatment. (B) TI-C cultures that were
passaged for several months were challenged with the indicated HDIs,
and GFP expression was quantitated by FACS at 24 h posttreatment.
con, not treated. Concentrations: dimethyl sulfoxide (DMSO), 0.05%
and 0.2%; TSA, 0.5, 1, and 2 ?M; apidicin, 0.5, 1, and 2 ?g/ml; valproic
acid (VPA), 2, 4, and 8 mM; sodium butyrate (NaBut), 2, 4, and 8 mM.
(C) Northern blot analysis of GFP mRNA was carried out using
standard methods. GFP mRNA was characterized in a pool of
GFP(?) HeLa cells infected with the ASV construct in which the GFP
gene is under control of the hCMV IE promoter (left panel). RNA
loading was monitored by staining of 18S and 28S RNAs, and these
species also served as sizing standards (5,025 and 1,868 nucleotides,
respectively). A GFP transcript of ca. 1,700 nucleotides was identified,
and this size is consistent with initiation within the internal hCMV viral
promoter and 3? processing at the 3? LTR. This transcript was not
detected in TI-C cells but was induced to significant levels after treat-
ment with TSA (1 ?M) (right panel).
VOL. 81, 2007 VARIEGATED REACTIVATION OF SILENT RETROVIRUSES2597
initially weak and broadly distributed, resulted in more uni-
form and intense GFP expression (Fig. 4, AP6, AP10, and
AP11). Northern blot analysis confirmed an increase in GFP
mRNA in GFP(?) cells following TSA treatment (not shown).
We concluded, therefore, that TSA treatment derepresses
GFP expression in GFP(?) cells. Although our qPCR analysis
indicated that the average GFP copy number in GFP(?) cells
was close to 1, we could not immediately exclude the possibility
that the increase in GFP expression was the result of activation
of secondary, silent GFP reporter genes that were present in
GPF(?) cells. However, we note that TSA treatment caused a
partial or complete shift in GFP intensity patterns from weak
and broad to more uniform and intense. As the distribution of
GFP intensity changes in response to TSA treatment, such
patterns are inconsistent with the activation of secondary, si-
lent GFP genes. To further address this issue, we used a linker-
mediated PCR-based strategy to amplify host-virus junctions
from one clone that displayed broad GFP expression (AP6);
only a single integrated viral DNA was detected (not shown).
TSA treatment of the representative GFP U clones, which
display more uniform and stronger GFP expression, produced
little change in GFP intensity (Fig. 4, AP2 and AP4). Further-
more, the small shifts in fluorescence intensities observed are
likely due to TSA-induced changes in cell morphology, as
treatment of uninfected HeLa cells with TSA produced a sim-
ilar shift in the autofluorescence signal (Fig. 4).
From the results described in Fig. 4 we conclude that in
GFP(?) U clones, GFP reporter expression is fully dere-
pressed. In contrast, in GFP(?) V clones, reporter expression
is subject to HDAC-mediated repression. We note that the
GFP intensity profiles in U clones are similar to the profiles
obtained after treatment of V clones with TSA (using the same
GFP intensity scale). Therefore, our analysis of the GFP(?) V
clones reveals a role for HDACs in epigenetic repression, and
analysis of the U clone identifies a situation in which the
reporter gene is highly resistant to HDAC-mediated repres-
sion (see Discussion and the model in Fig. 9). Although we
believe that these results are representative, we cannot rule out
the presence of multiple vector DNAs in some clones; multiple
integrations would produce a bias whereby high-level expres-
sion of GFP from one vector could obscure detection of a
weaker, variegated GFP phenotype produced by a second in-
Promoter-independent silencing of the GFP reporter gene.
The ASV-based vector described above utilizes the strong
hCMV IE promoter to drive GFP expression. To evaluate the
contribution of the promoter to the observed silencing phe-
nomena, we tested two other ASV vectors in which the GFP
reporter gene was driven either by the native LTR promoter or
a human cellular promoter derived from the EF-1 alpha gene.
The latter promoter is reported to drive persistent reporter
gene expression and has been exploited in vector design (21,
54). In the LTR-driven construct, the GFP gene is in the
position of the viral v-src gene and expressed through a spliced
mRNA, while the EF-1 alpha promoter replaces the internal
hCMV IE promoter. HeLa cells were infected with these vec-
tors under conditions that produced ca. 20% GFP(?) cells.
GFP(?) cells were sorted from infected populations and
treated with TSA. As observed previously with the hCMV
IE-driven GFP vector, GFP expression could be activated in a
subset of these GFP(?) cells. We then enriched for cells in
which GFP could be reactivated by TSA, and these populations
were designated TI-L and TI-E, corresponding to the LTR and
EF-1 alpha promoters, respectively. Rechallenge of these cells
with TSA resulted in robust GFP activation (Fig. 5), indicating
that the HDAC-mediated silencing that we have described for
ASV is not restricted to reporter gene expression that is initi-
ated from the hCMV IE promoter.
Reactivation of the HDAC-repressed GFP reporter gene by
prostratin is promoter specific. HIV-1 postintegration latency
is an epigenetic phenomenon. It has been demonstrated that
HDAC inhibitors can activate silent HIV, and such treatment
may be useful as part of a combined therapy to eliminate
latently infected cells (34, 36, 64). Prostratin is a phorbol ester
compound that has similar potential (35) and is able to activate
latent HIV-1 in cultured cells through stimulation of the
NF-?B pathway (Fig. 6A) (62). To identify factors or pathways
that might cooperate with HDACs to maintain silencing, we
FIG. 4. FACS analysis of GFP(?) cell clones after treatment with
TSA. Eleven GFP(?) clones were categorized as variegated (V) or
uniform (U) by microscopy. Analyses of several representative clones
(V clones, AP6, AP10, and AP11; U clones, AP2 and AP4) are pre-
sented. Cultures were treated with 0.5 ?M TSA for 24 h and analyzed
by FACS. Nonspecific effects were monitored by treatment of unin-
fected HeLa cells (HeLa). Data were processed with FlowJo software.
Untreated, no fill; TSA treated, filled.
2598KATZ ET AL.J. VIROL.
tested a variety of compounds for their abilities to reactivate
GFP expression in the TI-C and TI-L cultures. We found that
treatment with prostratin, as well as another phorbol ester,
PMA, resulted in robust GFP reactivation in the TI-C popu-
lation (Fig. 6B). Therefore, although these cells were selected
for HDAC-mediated silencing, direct inhibition of HDAC ac-
tivity per se is not necessary for reactivation; rather, stimula-
tion of the hCMV IE promoter with phorbol esters is appar-
ently sufficient (11). As prostratin stimulates the NF-?B
pathway (62), we hypothesized that the ability of this com-
pound to reactivate the silent GFP reporter gene was mediated
by NF-?B sites in the hCMV IE promoter. The ASV LTR does
not contain known NF-?B sites, and, consistent with the hy-
pothesis, treatment of TI-L cells with prostratin did not result
in reactivation of GFP (Fig. 6C). However, treatment of TI-C
cells with TNF-?, which stimulates the NF-?B pathway in
HeLa cells, failed to reactivate GFP expression (Fig. 6D). As a
positive control, we confirmed the ability of TNF-? to induce
HIV expression in the model cell system described for Fig. 6A,
as well as to trigger apoptosis in the TI-C culture in the pres-
ence of cycloheximide (data not shown). We conclude that the
abilities of prostratin and PMA to activate hCMV IE-driven
GFP expression in this system may be dependent on several
response elements in the promoter/enhancer (6, 11). These
results suggest that silent retroviral DNAs are accessible to the
general transcription machinery and that reactivation can be
mediated by local HAT recruitment and/or displacement of
Reactivation of an HDAC-repressed viral GFP reporter
gene may be dictated by switching between responsive and
nonresponsive states. Although the TI-C populations were
prepared by sorting cells in which GFP was activated in re-
sponse to TSA treatment, we noted routinely that challenge
with HDAC inhibitors or prostratin/PMA failed to reactivate
GFP expression in all cells in the population. Several micros-
copy-based experiments were designed to determine the basis
for this incomplete response. Single cells from the TI-C pop-
FIG. 5. Characterization of cell populations enriched for TSA-in-
ducible hCMV IE-, ASV LTR-, and EF1 alpha-driven GFP expression.
The indicated cell populations were treated with TSA (1 ?M) for 24 h,
and GFP expression was quantitated by FACS analysis. TI-C, hCMV
promoter; TI-L, ASV LTR promoter; TI-E, EF-1? promoter.
FIG. 6. Phorbol esters can reactivate silent GFP reporter gene expression in TSA-inducible TI-C but not TI-L populations. GFP expression in
treated and untreated (con) cells was quantitated by FACS analysis at 24 h posttreatment. (A) A Jurkat cell clone (10.6) harboring latent HIV
encoding a GFP reporter (30, 62) was treated with prostratin (2 ?M). (B) TI-C cells were treated with prostratin (0.1, 0.3, 1.0, and 1.5 ?M) or TSA
(2 ?M). (C) TI-L cells were treated with prostratin (0.3, 1.0, and 1.5 ?M) or TSA (2 ?M). (D) TI-C cells were treated with prostratin (2.0, 5.0
?M), PMA (100 and 200 nM), and TNF-? (10, 20 ng/ml).
VOL. 81, 2007 VARIEGATED REACTIVATION OF SILENT RETROVIRUSES2599
ulation were first plated at a high dilution, allowed to grow into
large colonies, and then challenged with prostratin or TSA. We
found that some cells within all of the colonies failed to reac-
tivate, leading to variegated patterns of GFP expression (not
shown). We hypothesized that this nonuniform response could
be due to differences in the metabolic state of cells in the
colony, nonclonal outgrowth (i.e., cross contamination of col-
onies), or switching between responsive and nonresponsive
To distinguish between these possibilities, we used an ap-
proach similar to that described above to follow variegation of
GFP(?) cells (Fig. 2). Mitotic shakeoff was performed on TI-C
cell cultures, and the cells were diluted and plated. After 1 to
2 h, the mitotic cells enter G1in a highly synchronous manner
(70 to 90%), with “colony birth” indicated by formation of cell
doublets (Fig. 2A). Synchronous growth could frequently be
followed to the four- and eight-cell stages. Cultures were then
treated with TSA (or prostratin) at various times postplating.
In this way, the reactivation patterns of individual cells in
synchronous, clonal populations could be mapped. As TSA
treatment causes HeLa cells to assume a spindle-like morphol-
ogy and detach from the plate more readily than untreated
cells, prostratin treatment was initially used to monitor pat-
terns of reactivation. TI-C colonies were treated with pros-
tratin ca. 16 h after colony birth, and GFP expression was then
monitored in microclones after ca. 48 h, when eight-cell colo-
nies were present (Fig. 7A). We observed a subset of GFP-
expressing colonies in which the GFP signal was present in four
out of the eight cells (variegated). To investigate this phenom-
enon further, TI-C colonies were treated with prostratin sev-
eral hours after colony birth, and GFP expression was then
monitored in microclones after ca. 24 h, when the majority had
reached the four-cell stage (Fig. 7B). A significant percentage
of colonies showed no response by microscopy, and this is
likely a reflection of the fact that this method of scoring GFP
expressing cells is less sensitive than FACS analysis. However,
among 38 four-cell colonies that contained GFP-expressing
cells, 12 colonies included four GFP-expressing cells (4/4, uni-
FIG. 7. Analysis of GFP reactivation patterns during clonal outgrowth of TI-C microclones reveals a variegated response. TI-C cell populations
were synchronized by mitotic shakeoff and plated under dilute conditions. Colonies were treated at various times postplating with prostratin, and
fluorescence (right) and phase-contrast (left) images were acquired as indicated in the diagrams: black fill, nonexpressing cells; gray fill,
GFP-expressing cells. (A) Treatment at the two-cell stage resulted in variegated (V) as well as uniform (U) GFP expression at the eight-cell stage.
Shown is a representative colony displaying variegated expression. (B) The two-cell colonies were treated with prostratin at 2 h postplating, and
colonies were imaged at the four-cell stage (boxed). Variegated and uniform expression patterns were observed. The top panel shows neighboring
V- and U-type colonies, and the bottom panel shows a V-type colony. Cell-to-cell intensity of GFP expression is constant within each clone (boxed).
(C) Treatment was similar to that for panel B, except that aphidicolin was included to prevent cells from progressing into S phase. Colonies were
arrested at the two-cell stage and could be quantitated according to V (1/2) or U (2/2) patterns (see Fig. 8). Representative images are shown.
2600KATZ ET AL. J. VIROL.
form), while 26 included only two GFP-expressing cells (2/4,
variegated). Representative images are shown in Fig. 7B. Strik-
ingly, none of the four-cell colonies contained either one or
three GFP-expressing cells. Furthermore, the intensities of the
two or four GFP-expressing cells within each colony were uni-
form and characteristic for each clone (Fig. 7B). As these cells
were obtained from a cell population, this pattern would be
consistent with a unique integration site in each microclone,
with the differences in GFP intensity between clones being due
to integration site position effects on expression. Overall, this
variegated reactivation pattern suggested a binary switch,
whereby cells could cycle between responsive and nonrespon-
sive states (see Discussion).
To test this idea further and in a more quantitative manner,
we asked what was the probability of a single cell giving rise to
one versus two responsive cells. We incorporated a cell cycle
arrest step, in which the G1cell doublets were treated with
prostratin plus aphidicolin to prevent entry into S phase and
further cell divisions. This protocol resulted in persistence of
G1cell doublets that could be scored for one out of two (1/2,
variegated) or two out of two (2/2, uniform) cells in which GFP
expression was reactivated (Fig. 7C). The results showed a
distribution of 1/2 (variegated) and 2/2 (uniform) GFP(?) cell
doublets among those that expressed GFP (Fig. 8A). These
patterns were maintained at 24 and 48 h postplating, indicating
that GFP reactivation was not simply lagging in one cell in the
variegated doublets. This general pattern could be reproduced
using TSA with both the TI-C and TI-L cells, indicating that
this switching effect is not dependent on the CMV promoter or
the chemical inducer (Fig. 8B). The frequency of this asym-
metric response is much higher that than that expected for
chromosome loss; furthermore, loss of the GFP gene at the
observed rate would result in a severe and cumulative loss of
response to inducers in the population, which we have not
observed. To directly distinguish between loss of the GFP gene
and epigenetic effects, we allowed colonies to grow to a large
size (30 to 50 cells) before treatment with TSA or prostratin.
We typically observed several sectors of GFP reactivation in
these colonies, suggesting that the inability to respond to in-
ducers was mediated by reversible epigenetic restrictions.
From these experiments, we conclude that reactivation can
be restricted by additional epigenetic mechanisms. Further-
more, as cells were plated in M phase and aphidicolin treat-
ment blocked entry into S phase, these microclone experiments
established that reactivation could occur entirely within G1.
Integration of retroviral DNA establishes a stable associa-
tion with the host genome but does not ensure viral gene
expression. Over the last two decades, numerous studies have
addressed the role of viral and host determinants in retroviral
epigenetic silencing, with major focus on the behavior of ret-
rovirus-based vectors in stem cells. Here we describe high-
frequency HDAC-mediated epigenetic silencing and repres-
sion of an ASV-based vector in HeLa cells. We believe that this
behavior reflects specific host-virus interactions, as we found
that the hCMV IE GFP cassette is highly resistant to silencing
and repression when introduced into HeLa cells with an HIV-
1-based vector (Fig. 1 and data not shown).
Based on our current and previous findings, we can outline
several ways in which host-virus interactions could influence
silencing initiation, maintenance, and reactivation (Fig. 9). Al-
though we have observed variations in GFP reporter gene
expression that could be attributed to position affects and po-
sitional variegation, we also found that a large fraction of
integrated viral DNAs are subject to epigenetic repression or
silencing, as indicated by the response to HDIs (Fig. 9A). This
feature is illustrated by the finding that GFP intensities in some
GFP(?) cell clones increased and became more uniform after
TSA treatment and that a large fraction of GFP(?) cells har-
bor silent, TSA-inducible viral GFP reporter genes. In con-
trast, some GFP(?) clones displayed more intense and uni-
form GFP expression which was unaffected by TSA treatment.
These findings suggest the existence of a broad continuum of
HDAC-mediated epigenetic effects of varying severity (Fig.
9A). The pervasiveness of the HDAC-mediated repression and
silencing in this ASV system suggests that a particular cue
(cis-acting viral signal or cell-virus interaction) may trigger this
We and others have previously characterized the integration
site selection preferences for ASV DNA in HeLa cells and
have found that protein-coding genes are preferred modestly
as integration targets (4, 47). As a large fraction of integrated
genomes are subject to either epigenetic repression or silenc-
ing, it is unlikely that the integration site locus is the sole
determinant of the epigenetic fate of the integrated DNA.
Furthermore, we have found that integration site selection
patterns in GFP(?) and GFP silent TI cell populations are
similar (not shown). These findings disfavor a model in which
repression and silencing are due to integration in or near
constitutive heterochromatin. It will be important to charac-
FIG. 8. Quantitation of variegated (V) and uniform (U) GFP ex-
pression in two-cell colonies after aphidicolin arrest and reactivation
with prostratin (1 ?M) or TSA (0.5 ?M). The experimental design is
shown in Fig. 7C, and representative results are shown. (A) TI-C 2-cell
colonies were quantitated for V or U patterns at 24 and 48 h after
plating and prostratin treatment. (B) Comparison of TI-C and TI-L
colonies induced with TSA. The sample numbers are lower with TI-L
cells, as LTR-driven GFP expression is generally weaker and thus
more difficult to detect by microscopy.
VOL. 81, 2007 VARIEGATED REACTIVATION OF SILENT RETROVIRUSES2601
terize integration sites and GFP expression in individual cell
clones to substantiate this interpretation. Overall, the high-
frequency and TSA-sensitive repression and silencing de-
scribed here are consistent with our previous findings using
chromatin immunoprecipitation, which showed that ASV
DNA complexes associate with the transcriptional repressor
Daxx and HDACs soon after infection (22) (Fig. 9B). Daxx is
known to interact with HDACs (26) and is therefore an attrac-
tive candidate for initiating these events.
Based on our collective findings and the interpretations dis-
cussed above, our current model is that HDACs associate with
ASV DNA as part of an antiviral response (22). HDACs have
also been implicated as part of an intrinsic cellular response
that inhibits hCMV infection (52, 55), and HDIs have been
shown to stimulate expression of a variety of viral genomes (38,
43, 46, 67). Interestingly, the avian adenovirus protein Gam1
binds to and inhibits HDAC1 and may represent a virus-en-
coded HDAC inhibitor that serves to counter the antiviral
effects of HDACs (10, 13, 38). Similarly, the hCMV pp71
protein apparently acts to relieve Daxx-mediated transcrip-
tional repression of hCMV gene expression (5, 52, 55).
A correlation between DNA methylation and retroviral si-
lencing is well established, and some studies have provided
strong evidence for a causal relationship (58, 59). We found
that in our experimental system, HDAC inhibitors are suffi-
cient to reactivate silent viral DNA in a promoter-independent
fashion, and the DNA methyltransferase inhibitor 5-azacyti-
dine has only marginal and temporal-specific effects (data not
shown). Thus, the phenomenon that we have described ap-
pears to be an example of DNA methylation-independent,
HDAC-mediated silencing (50). We hypothesize that the re-
pressive action of HDACs is mediated through removal of
acetyl groups of histone tails, minimally within the silent pro-
moter regions, as has been demonstrated previously for the
hCMV promoter (8). However, we cannot rule out that reac-
tivation by HDIs is mediated through an indirect mechanism.
Our studies have also shown that the phorbol esters pros-
tratin and PMA can reactivate the silenced GFP reporter gene
and that this effect is promoter specific (i.e., occurs in TI-C but
not TI-L cells). The hCMV IE promoter can be activated by
phorbol esters, likely through stimulation of several transcrip-
tion factors that target this promoter (6). These transcription
factors may cause dissociation of HDACs and/or promote re-
cruitment of HAT coactivator complexes (Fig. 9B). Our results
suggest that reactivation does not require global inhibition of
HDAC activities but rather that local, promoter-specific an-
tagonism of HDACs is sufficient (Fig. 9B) and that the inte-
grated vector DNAs can be accessible to transcription factors.
These results are incompatible with a model in which silencing
is driven by integration into inaccessible heterochromatin.
A major and novel finding of this study relates to the mech-
anism of reactivation from silencing. We used a sorting strat-
egy to enrich for a cell population in which the GFP reporter
gene was silent but could be reactivated by TSA (TI cells).
Despite this selection, we found that rechallenge of asynchro-
nous TI cell populations with TSA induced GFP expression in
only a subset of cells. By following reactivation in synchronous
microclones, we found that variegated patterns of GFP reac-
tivation, where detected, appeared to be due to orderly
“switching” between HDI-responsive and nonresponsive states
(Fig. 9C). The time scale of our experiments (e.g., four-cell
colonies) apparently favors the detection of a single switching
event, manifested as 2/4 GFP-expressing cells. Analysis of
eight-cell colonies occasionally revealed a 2/8 GFP(?) sec-
tored pattern, suggesting that “switching” occurred during the
second synchronous division. Furthermore, treatment of larger
colonies resulted in a sectored pattern featuring clusters of
GFP(?) cells (not shown).
One interpretation of these results is that a dominant, epi-
genetic phenomenon, which imparts HDI resistance, mediates
this variegated response. We speculate that this switching is a
manifestation of temporal variations in transcriptional compe-
tence of the integrated DNA, as has been observed for cellular
genes and introduced reporter genes (2, 48, 53), and such
effects may be more readily observed in our system due to the
fact that the viral locus is haploid. In support of this interpre-
tation, we note that the characteristic variegated patterns of
drug-induced GFP expression in microclones are similar to the
FIG. 9. Models for epigenetic silencing and repression. (A) Model
for a continuum of HDAC-mediated repression and silencing in
GFP(?) and GFP(?) cells. The model is based on the broad GFP
expression profiles and the stimulation of GFP expression in both
GFP(?) and GFP(?) variegated (V) clones by TSA. Filled arrow-
heads indicate strong (large arrowhead) or weak (smaller arrowheads)
HDAC effects that are modulated by the integration site. (B) Model
showing interaction between Daxx/HDAC and integrated retroviral
DNA (22). TI cells were selected for the ability of the GFP reporter
gene to be reactivated by TSA, an HDI. In such selected cells, phorbol
esters can also reactivate the GFP reporter gene, suggesting that the
silent loci can be available to transcription factors (rectangle). Co-
activator complexes containing HATs may overcome or displace
HDACs. (C) The model indicates a common mechanism underlying
two phenomena: variegation in some GFP(?) clones and variegated
reactivation in GFP(?) TI cells. Expression could be restricted in one
daughter cell due to transient inaccessibility of the integration site
locus to transcriptional centers (48).
2602KATZ ET AL. J. VIROL.
patterns of GFP expression observed during outgrowth of
some GFP(?) microclones (e.g., compare Fig. 2B left middle
panel and 7A; Fig. 9C). Taken together, the patterns suggest
that depending on the integration site, the viral loci may be
only transiently accessible for transcription (2, 48, 53), result-
ing in variegated expression. In some GFP(?) TI cell micro-
clones, interrogation with the HDIs or activators reveals this
variegated response, which we interpret as being the result of
underlying “on-off switching” of transcriptional competence;
GFP sectoring after challenge would be expected if such states
are epigenetically heritable over several cell divisions. We sug-
gest, therefore, that transient, locus-specific transcriptional in-
accessibility accounts for both the variegated GFP reactivation
observed after HDI or phorbol ester treatment and the varie-
gated expression in GFP(?) colonies. Overall, these studies
have apparently uncovered an important variable that can con-
tribute to the effectiveness of HDAC inhibitors. Furthermore,
our results may explain the transcriptional oscillations of some
viral reporter genes, as well as the nonuniform reactivation of
silent retroviral DNA observed in other systems (20, 62). In the
course of these microscopy-based studies, we found that reac-
tivation could occur in synchronized cells that were arrested at
the G1-S border of the cell cycle (Fig. 7C). We have confirmed
this result using FACS analysis (data not shown). This finding
suggests that reactivation can occur through local HAT-medi-
ated acetylation of preexisting nucleosomes, rather than
through DNA replication-coupled deposition of acetylated hi-
In summary, we report high-frequency epigenetic silencing
and repression of retroviral DNA expression. Our studies re-
veal that such silencing is rapid and is likely mediated by
HDACs that associate with viral DNA early after infection.
The interpretation of responses to chemical treatments sug-
gests that reactivation can occur either through inhibition of
HDACs or by local recruitment of transcriptional activators.
Overall, our results suggest that such silencing and repression
can occur independently of the integration site but that the
location can influence the degree of repressive effects as well as
the ability to respond to reactivating stimuli.
This work was supported by National Institutes of Health grants
CA71515 and CA06927 and also by an appropriation from the Com-
monwealth of Pennsylvania. Support for R.A.K. was funded in part
under a grant with the Pennsylvania Department of Health.
The following Fox Chase Cancer Center Shared Facilities were used
in the course of this work: the Automated DNA Sequencing Facility,
the Flow Cytometry and Cell Sorting Facility, and The Fannie E.
Rippel Biochemistry and Biotechnology Facility.
We thank Joe Ramcharan for providing the ASV-EF-1 alpha GFP
vector and Yanacha Toporovskaya, Elizabeth Farley, Kim Boland, and
Paul Garr for technical assistance. We also thank Konstantin Taganov
for analyses of integrated ASV-GFP vectors and helpful discussions.
We are also grateful to Eric Verdin for providing HIV-GFP-infected
Jurkat cell clones. We thank Severin Gudima and Jared Evans for
critical comments on the manuscript and Marie Estes for assistance in
preparing the document.
The contents of this paper are solely the responsibility of the authors
and do not necessarily represent the official views of the National
Cancer Institute or any other sponsoring organization. The Pennsyl-
vania Department of Health specifically disclaims responsibility for any
analyses, interpretations, or conclusions.
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2604KATZ ET AL.J. VIROL.