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Previous Article | Next Article 
Journal of Virology, May 2001, p. 4482-4489, Vol. 75, No. 10
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4482-4489.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cell Cycle Analysis of Epstein-Barr Virus-Infected
Cells following Treatment with Lytic Cycle-Inducing Agents
Antonio
Rodriguez,
Eun Joo
Jung, and
Erik K.
Flemington*
Department of Pathology, Tulane Cancer
Center, New Orleans, Louisiana 70112
Received 7 November 2000/Accepted 2 February 2001
 |
ABSTRACT |
While Epstein-Barr virus (EBV) latency-associated gene expression
is associated with cell cycle progression, the relationship between the
EBV lytic program and the cell cycle is less clear. Using four
different EBV lytic induction systems, we address the relationship
between lytic cycle activation and the cell cycle. In three of these
systems, G0 or G1 cell growth arrest signaling is observed prior to detection of the EBV immediate-early gene product
Zta. In tetradecanoyl phorbol acetate-treated P3HR1 cultures and in
5-iodo-2'-deoxyuridine-treated NPC-KT cultures, cell cycle analysis of
Zta-expressing cell populations showed a significant G1
bias during the early stages of lytic cycle progression. In contrast,
treatment of the cell line Akata with anti-immunoglobulin (Ig) results
in rapid induction of immediate-early gene expression, and accordingly,
activation of the immediate-early gene product Zta precedes significant
anti-Ig-induced cell cycle effects. Nevertheless, cell cycle analysis
of the Zta-expressing population following anti-Ig treatment shows a
bias for cells in G1, indicating that anti-Ig-mediated
induction of Zta occurs more efficiently in cells traversing
G1. Last, although 5-azacytidine treatment of Rael cells
results in a G1 arrest in the total cell population which precedes the induction of Zta, cell cycle analysis of the
Zta-expressing population shows a significant bias for cells with an
apparent G2/M DNA content. This bias may result, in part,
from activation of Zta expression following demethylation of the Zta
promoter during S-phase. Together, these studies indicate that
induction of Zta occurs through several distinct mechanisms, some of
which may involve checkpoint signaling.
| |
INTRODUCTION |
Epstein-Barr virus (EBV) is
associated with a variety of cancers in humans including African
Burkitt's lymphoma, Hodgkin's disease, and nasopharyngeal carcinoma
(16, 21, 28), and recent studies have identified EBV in
breast tumors (3, 18, 19). EBV is also a causative agent
in lymphoproliferative disorders in immunocompromised individuals
(16, 21).
EBV utilizes two separate classes of genes that carry out very distinct
functions in its life cycle (16, 17). The latency-type gene expression patterns are associated with cell proliferation, and
many of these genes function, in part, to activate cell cycle pathways
leading to cell proliferation. Accordingly, some form of latency gene
expression is invariably observed in EBV-associated tumors. Although
the association between latency gene expression and cell cycle
progression is well established, the relationship between the lytic
gene expression program and the cell cycle is less well understood.
Nevertheless, previous studies have provided evidence that in contrast
to the latency gene expression program, the lytic replication cycle may
be associated with a nonproliferating cellular milieu. In the oral
epithelium, lytic replication occurs primarily in the outer, more
differentiated layers (2, 26, 27). Other studies have
shown that the immediate-early EBV gene product Zta can induce cell
growth arrest (5-7, 22), indicating that the EBV lytic
program has evolved a mechanism to shut down cellular DNA synthesis.
Cell cycle regulation during activation and progression of the lytic
cascade, however, has received only limited attention (5,
12). Here we report an examination of cell cycle alterations
associated with induction and progression of EBV lytic replication in
four distinct lytic cycle induction systems.
| |
MATERIALS AND METHODS |
Cell culture.
NPC-KT cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum
(Life Technologies), 2 mM glutamine, 100 µg of streptomycin per ml,
and 100 U of penicillin per ml in a humidified atmosphere at 37°C
with 5% CO2-95% air. The Burkitt's lymphoma cell lines
Rael, Akata, and P3HR1 were cultured as indicated above except that
they were propagated in RPMI medium supplemented with 10% fetal bovine
serum, 2 mM glutamine, streptomycin (100 µg/ml), and penicillin (100 U/ml). Induction experiments were carried out as indicated in the
figure legends.
Cell cycle analysis.
For cell analysis of whole-cell
populations, cells were collected, washed once with 1×
phosphate-buffered saline (PBS), suspended in 5 ml of cold (4°C) 1×
PBS-0.1% glucose, fixed with 5 ml of 70% cold (
20°C) ethanol for
at least 45 min at 4°C, washed with 1× PBS, and treated for 45 min
at 37°C with RNase A (0.1 mg/ml) in 69 mM propidium iodide-38 mM
sodium citrate. Cell cycle analysis was carried out with a FACScan
fluorescence-activated cell sorter (FACS) (Becton Dickinson).
Cell cycle analysis of Zta selected cells was carried out as follows.
Induction experiments were performed as indicated in the figure
legends. At the indicated time points, cells were collected, washed
once with 1× PBS, suspended in 0.5 ml of cold (4°C) 1× PBS-0.1%
glucose, and fixed with 5 ml of 70% cold (20°C) ethanol for at
least 45 min at 4°C (and up to 1 week). Fixed cells were spun down
and washed one time with 5 ml of 1× PBS. Cells were then suspended in
50 to 100 µl of 1× PBS. One milliliter of 3.7% formaldehyde was
added, and the samples were mixed briefly and then incubated for 10 min
with gentle rocking. The cells were diluted with 10 ml of 1× PBS, spun
down, and washed two times with 1× PBS. Samples were then suspended in
1 ml of 0.2% Triton X-100 and incubated for 3 min. Then 10 ml of 1×
PBS was added, and the cells were spun down and washed one time with 10 ml of 1× PBS. After taking off the supernatant, cells were spun
briefly, and the remaining PBS was taken off. Cells were then suspended in 50 µl of the first antibody solution containing a 1:50 dilution of
anti-Zta polyclonal antibody RR839 (a generous gift from George Miller)
and 10% fetal bovine serum in 1× PBS. The samples were incubated for
1 h at room temperature with occasional mixing. Then 1 ml of 1×
PBS was added, and cells were spun down. Cells were then suspended in
200 µl of second antibody solution containing a 1:50 dilution of
fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin
(Ig) antibody and 10% fetal bovine serum in 1× PBS. Samples were then
incubated for 30 min at room temperature in the dark. The cells were
washed once with 1 ml of 1× PBS and suspended in 300 µl of 69 mM
propidium iodide-38 mM sodium citrate solution, and RNase A was added
to a final concentration of 0.1 ml/ml. Cells were incubated for 30 to
60 min at 37°C and then analyzed by fluorescence-activated cell sorting.
Western blot analysis.
After a single 1× PBS wash, a
fraction of cells harvested for cell cycle analysis were separated for
Western blot analysis. Cells were immediately suspended in 15 pellet
volumes of sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) loading buffer (Laemmli) (21) and boiled for 20 min to shear the DNA. Cell lysates were subjected to SDS-PAGE
separation and transferred to nitrocellulose membranes. The blots were
blocked for 5 to 30 min in blocking buffer (Tris-buffered saline, 0.5% Tween 20 [TBST] containing 5% lowfat powdered milk and 1% fetal bovine serum) and then incubated with the indicated primary antibody (in blocking buffer) overnight at 4°C. The blots were washed three times with 1× TBST for 15 min each. The blots were then incubated with
peroxidase-conjugated secondary antibody in blocking buffer for 1 h at room temperature. Blots were washed as above and analyzed with an
enhanced chemiluminescence detection system (Amersham) according to the
manufacturer's recommendations, and filters were exposed to Kodak XR
film. Antibodies employed for each experiment are indicated in the
figure legends.
| |
RESULTS |
Cell cycle alterations in 5-iodo-2'-deoxyuridine-treated NPC-KT
cells.
Initiation of the lytic cycle in NPC-KT cells by
transfecting with a plasmid that constitutively expresses the
immediate-early gene product Zta results in cell growth arrest in the
G0/G1 phase of the cell cycle (5,
8). In this system, growth arrest is accompanied by induction of
the tumor suppressor protein p53 as well as the cyclin-dependent kinase
inhibitors p27 and p21 (5, 6; unpublished data). To extend
these studies, we analyzed alterations in the cell cycle control
machinery in NPC-KT cells treated with the exogenous lytic
cycle-inducing agent 5-iodo-2'-deoxyuridine. As shown in Fig.
1A, addition of 5-iodo-2'-deoxyuridine to
NPC-KT cell cultures causes delayed induction of Zta at 48 and 72 h. Induction of Zta expression coincides with an increase in the percentage of cells in the G0/G1 phase of the
cell cycle (Fig. 1B), suggesting a possible relationship between these
two events. Notably, there is a substantial and reproducible decrease
in the G0/G1 population (and a corresponding
increase in the G2/M population) at 16 h, although we
do not at present understand its significance. At the molecular level,
several changes in cell cycle control proteins occur prior to any
detectable expression of Zta (downregulation of c-Myc and induction of
p53, p27, and p21). This suggests that although Zta expression
correlates temporally with the onset of growth arrest in this induction
system, the molecular changes that elicit growth arrest may be upstream
from Zta signaling. Although we have shown previously that Zta can
induce growth arrest, the data suggest the possibility that Zta
expression may itself be influenced by growth arrest signaling.
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FIG. 1.
Lytic cycle induced in NPC-KT cells by treatment with 75 µg of 5-iodo-2'-deoxyuridine (IDU) per ml. Cells were harvested at
the indicated times, and a portion of the cells were lysed immediately
in SDS-PAGE loading buffer, boiled for 20 min, and loaded onto an
SDS-PAGE gel for Western blot analysis (A). The remaining fraction of
cells were analyzed for cell cycle distribution by FACS analysis of
propidium iodide-stained cells (B). Zta was detected using the M47
polyclonal antibody, and p53, p27, and p21 were detected using the DO-1
(Santa Cruz Biotechnology), anti-Kip1/p27 (Transduction Laboratories),
and anti-Cip1/p21 (Transduction Laboratories) monoclonal antibodies,
respectively.
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|
In most EBV lytic cycle induction systems, only a fraction of the cell
population typically enters the lytic cascade. Although treatment of
NPC-KT cells with 5-iodo-2'-deoxyuridine leads to an increase in the
percentage of cells in the G0/G1 phase of the cell cycle, it was important to determine whether a similar disposition occurred specifically in the cell population undergoing lytic cycle
progression. To address this issue, NPC-KT cells were induced with
5-iodo-2'-deoxyuridine and the DNA content was determined specifically
in the Zta-expressing cells. Importantly, previous studies in other
herpesvirus systems have shown that viral replication can measurably
influence the DNA content and lead to mistaken conclusion that cells
are in S or G2/M (4, 13). In an attempt to
avoid this potential complication, we assayed the cell cycle distribution in Zta-positive cells at early time points following the
first detection of Zta. As shown in Fig.
2, at 38 and 48 h post-5-iodo-2'-deoxyuridine treatment, both the Zta-positive population and the total population are enriched for G0/G1
cells. This indicates that initiation of the EBV lytic cascade occurs
in a G0/G1-enriched cell population in this
system.
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FIG. 2.
Cell cycle distribution of NPC-KT cells following
exposure to 75 µg of 5-iodo-2'-deoxyuridine (IDU) per ml was analyzed
by FACS analysis of propidium iodide-stained cells. Zta-positive cells
were selected using the polyclonal anti-Zta antibody RR839 (a generous
gift from George Miller). The cell cycle experiment shown here has been
repeated with similar results.
|
|
Cell cycle alterations following TPA treatment of Burkitt's
lymphoma cell line P3HR1.
To further address the relationship
between lytic replication and the cell cycle, we extended our study to
analyze the cell cycle in B-cell lytic induction systems. As shown in
Fig. 3, treatment of the Burkitt's
lymphoma cell line P3HR1 with the phorbol ester tetradecanoyl phorbol
acetate (TPA) results in a significant enrichment of cells in the
G0/G1 phase of the cell cycle within 24 h
of treatment (note: a portion of these G0/G1
cells appear to reenter the cell cycle at 48 and 72 h). Although
Zta expression is induced slightly at 24 h (Fig. 3B), significant
expression of Zta is not observed until the 48-h time point, indicating
that growth arrest largely precedes induction of Zta in TPA-treated
P3HR1 cells.
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FIG. 3.
P3HR1 cells were treated with TPA (20 ng/ml) for the
indicated times. Cells were harvested and analyzed for cell cycle
distribution (A) and protein expression (B) as described in the legend
to Fig. 1.
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The TPA-induced G0/G1 arrest in P3HR1 cells
appears to occur through a mechanism that is distinct from the growth
arrest elicited by 5-iodo-2'-deoxyuridine in NPC-KT cells. At 24 h
following addition of TPA, when growth arrest is maximal, no changes in
p21, p27, or p53 are observed (Fig. 3B). In addition, P3HR1 cells
harbor a mutant p53, further discounting a likely role for p53 in
TPA-mediated growth arrest. Interestingly, although p27 levels are not
altered at 24 h, there is an apparent delayed induction at 48 and
72 h. We have previously shown that Zta induces p27 in epithelial
systems (5), and the temporal correlation between p27 and
Zta induction suggests that Zta may play a role in the activation of
p27 expression in this system.
As shown in Fig. 4, we also analyzed the
cell cycle distribution in the Zta-positive population following TPA
treatment of P3HR1 cells. Like the whole cell population, an enrichment
of G0/G1 cells is also observed in the
Zta-expressing population at 24 and 38 h (Fig. 4). Notably, fewer
Zta-expressing cells reenter the cell cycle at 38 h compared to
the percentage of the total population that reenter the cell cycle at
this time point (Fig. 4). It is possible that this results from
Zta-mediated growth arrest functions, which might involve p27, or from
the action of some other EBV-encoded lytic gene product. Further
experiments will be required, however, to adequately address this
issue.
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FIG. 4.
Cell cycle analysis of the whole population and
Zta-positive cells following treatment of P3HR1 cells with TPA (20 ng/ml). Zta-positive cells were selected using the polyclonal anti-Zta
antibody RR839. The cell cycle experiment shown here has been repeated
with similar results.
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Cell cycle regulation in anti-Ig-treated Akata cells.
Unlike
5-iodo-2'-deoxyuridine stimulation of NPC-KT cells and TPA stimulation
of P3HR1 cells, activation of the lytic cascade through anti-Ig
treatment of Akata cells has been shown previously to occur with rapid
kinetics, with significant Zta induction typically observed within
4 h (25). This rapid response likely precludes the
involvement of a growth arrest response as a significant factor in
lytic cycle activation in this system, since such cell synchronization requires a considerably longer time frame. As shown in Fig.
5, treatment of Akata cells with anti-Ig
results in induction of Zta between 4 and 8 h posttreatment and no
apparent G0/G1 arrest is observed even through
48 h postinduction. These data indicate that, as expected, anti-Ig
signaling of Zta expression likely occurs through a more direct
mechanism.
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FIG. 5.
Akata cells were treated with anti-Ig (1 mg/ml) for the
indicated times and cells were analyzed for cell cycle (A) and protein
expression (B) as described in the legend to Fig. 1.
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The p53 gene has been shown previously to be deleted in Akata cells
(1), and accordingly, no p53 protein was detected by Western blot analysis (data not shown). Analysis of p27 levels showed a
gradual decrease between 0 and 48 h (Fig. 5B), indicating that
anti-Ig treatment does not elicit p27 induction. This result also
suggests either that Zta cannot activate p27 expression in this cell
system or that anti-Ig treatment downregulates p27 expression and Zta
cannot overcome this activity. Analysis of p21 expression, on the other
hand, revealed a transient induction at 2 and 4 h post-anti-Ig
treatment (Fig. 5B), and although growth arrest is not involved in
signaling Zta expression in this system, it remains possible that this
induction of p21 may in some way be involved in lytic cycle signaling.
We also analyzed the cell cycle distribution in the Zta-positive cell
population to determine whether there might be any virus specific cell
cycle effects. As shown in Fig. 6, an
enrichment of cells in G0/G1 was observed in
the Zta-positive population relative to the whole population at 4, 6, and 8 h post-anti-Ig treatment. Similarly, there is an increase in the
percentage of G2/M-phase cells in the Zta-positive
population at 4, 6, 8, and 10 h. It is unlikely that this
enrichment is the result of the activity of any lytic gene expression
(such as Zta) on the cell cycle control machinery, since it occurs so
rapidly. Instead, it is more likely that although anti-Ig treatment may
activate Zta expression through a relatively direct mechanism that does not require the induction of cell growth arrest, it may nevertheless cooperate with a G1- and G2/M-specific
factor(s) to fully activate Zta expression. Alternatively, it is also
possible that there is an S-phase-specific factor that inhibits Zta
expression.
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FIG. 6.
Lytic cycle induced in Akata cells by incubating with
anti-Ig (1 mg/ml) for the indicated times. Cell cycle distribution was
analyzed in the total cell population and the Zta-positive population
using the anti-Zta antibody RR839.
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As shown in Fig. 6, anti-Ig treatment of Akata cells results in a
significant increase in the number of cells with less than a
2n DNA content. Interestingly, this activity is strongly
suppressed in the Zta-positive cell population, suggesting that a viral
lytic antigen(s) functions to block the apoptotic and/or necrotic
anti-Ig response. Suppression of this activity is observed at the
earliest time point that Zta is detected, indicating that this viral
activity must be encoded by a very early gene. Similar results have
been observed by another group, who showed that an EBV lytic gene
specifically inhibits apoptosis induced by lytic cycle-inducing agents
(G. Inman, U. Binne, G. Parker, P. Farrell, and M. Allday, submitted for publication).
Cell cycle analysis of Rael cells following treatment with
5-azacytidine.
As shown in Fig. 7,
induction of the lytic cycle in the Burkitt's lymphoma cell line Rael
by treatment with 5-azacytidine results in an increase in the
percentage of cells in G1 in the whole cell population. The
enrichment of G0/G1 cells is first observed at
12 h and is pronounced by 24 h. In this time course experiment, Zta is not detected until 24 h, suggesting that growth arrest occurs either prior to or coincident with Zta expression. Unexpectedly, however, analysis of the cell cycle distribution specifically in Zta-positive cells showed a strong enrichment for cells
in G2/M (Fig. 8). As
mentioned above, studies in other herpesvirus systems have shown that
viral replication can make a significant contribution to the total DNA
content, leading to the mistaken impression that cells undergoing lytic
replication are in the S or G2/M phase of the cell cycle
(4, 13). In an effort to avoid the possible contribution
of newly replicated viral DNA in the cell cycle analysis, we carried
out a more detailed time course so that we could specifically analyze
the cell cycle distribution of Zta-positive cells at the earliest
possible time points. As shown in Fig. 9,
analysis of Zta-positive cells at the earliest time that Zta is
detected showed a strong bias for cells in G2/M. To further
address whether this G2/M enrichment is the result of
possible viral replication, we carried out an induction experiment in
the presence of the viral replication inhibitor foscarnet. The presence
of foscarnet, however, did not influence the G2/M bias of
Zta-positive cells (data not shown). Lastly, with DNA samples prepared
from the experiment shown in Fig. 9, no detectable viral DNA
replication was observed even at the 28-h time point (data not shown).
Together, these results indicate that the enrichment of cells with an
apparent 4n DNA content specifically in the Zta-positive
population is not due to viral replication.
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FIG. 7.
Rael cells were incubated with 10 µM 5-azacytidine
(5-AZA), and cells were analyzed for cell cycle distribution at the
indicated times. Anti-Zta Western blot was performed as described in
the legend to Fig. 1.
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FIG. 8.
Rael cells were incubated with 10 µM 5-azacytidine
(5-AzaC), and cells were analyzed for cell cycle distribution at the
indicated times. Cell cycle analysis of Zta-positive cells was carried
out using the anti-Zta antibody RR839. Pop., population.
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FIG. 9.
Cell cycle analysis (A) of uninduced and induced whole
populations (Pop.) and Zta-positive populations as described in the
legend to Fig. 8. Western blot analysis was performed as described in
the legend to Fig. 1.
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Like the results shown above for TPA-treated P3HR1 cells, no
significant change in p53 levels is observed in 5-azacytidine-treated Rael cells, and this is consistent with the mutant status of p53 in
Rael cells (Fig. 9B). On the other hand, a detectable increase in p27
is observed at 12 h following the addition of 5-azacytidine, and
this early induction may be the result of the treatment itself. A
further increase is observed around 16 to 20 h, which may also be
related to the treatment itself and/or the onset of Zta expression, which is first observed between 16 and 18 h (Fig. 9A and B). No p21 was
detectable at any time, which may be due to a lack of induction or to
low basal levels.
| |
DISCUSSION |
It has become clear over the last few years that unlike small DNA
tumor viruses, herpesviruses have likely evolved to replicate their DNA
in arrested or nonproliferating tissues (10). Moreover, studies from a number of laboratories have shown that herpes simplex virus (HSV), cytomegalovirus (CMV), and EBV all encode factors that
interact with the cell cycle control machinery in a manner that
activates certain cell cycle checkpoint functions (10). Although activation of these checkpoints can, in some settings, lead to
an apparent G2/M arrest, the accumulating literature
suggests that these herpesviruses likely evolved to elicit
predominantly a G0 and/or G1 growth arrest.
We have shown previously (5) that Zta activates a
G0/G1 checkpoint (although in certain
EBV-negative settings, Zta can also induce a G2/M
checkpoint response [unpublished]), supporting the notion that EBV
has evolved to carry out its viral replication cycle in the
G0/G1 phase of the cell cycle. Here we show
that in several lytic cycle induction systems, initiation of the lytic cascade occurs preferentially in G0/G1 cell
populations. Cell cycle analysis of Zta-positive cells from TPA-induced
P3HR1 cells, 5-iodo-2'-deoxyuridine-treated NPC-KT cells, and
anti-Ig-stimulated Akata cells showed an enrichment for cells in
G0 or G1.
TPA treatment of P3HR1 cells and 5-iodo-2'-deoxyuridine stimulation of
NPC-KT cells resulted in G0/G1 growth arrest
signaling prior to detectable expression of Zta. Furthermore, there is
a significant delay of at least 24 h before Zta expression is
induced in both of these systems, which is approximately the time
required for significant cell cycle synchronization. It is clear that
this delay is not an intrinsic property of the virus, since Zta
expression occurs with rapid kinetics in anti-Ig-treated Akata cells.
Together, these data suggest the possibility that some signaling events that are specifically activated in growth-arrested cells may be involved in inducing Zta expression. As such, EBV may have evolved with
this response mechanism to detect when cells are in G0
and/or G1 so that the virus can initiate the lytic cascade
under conditions that favor efficient progression of lytic replication.
Signaling of the lytic cycle by anti-Ig, on the other hand, appears to
occur through a distinct mechanism that more directly activates Zta expression. Nevertheless, cell cycle analysis of the Zta selected population revealed a bias for G1 (and G2/M)
cells, suggesting that although anti-Ig signaling may not directly
involve growth arrest, it may cooperate with G1-specific
factors to activate Zta expression.
Another group has recently reported that induction of the lytic cycle
in EBV-infected B lymphocytes through stimulation of CD40 similarly
leads to concomitant growth arrest and induction of p27
(12), further supporting the idea that the Zta promoter may be responsive to growth arrest signaling. In other studies, analysis of Zta's promoter has identified elements that have been found to be binding sites for the terminal differentiation factor MEF2
(11, 24), and Karimi et al. (15) identified a
region that is responsive to epithelial cell differentiation.
Therefore, accumulating evidence suggests that the Zta promoter is
specifically responsive to growth arrest and/or terminal
differentiation signaling.
Immediate-early gene expression in CMV and HSV has similarly been shown
to be regulated in the G1 phase of the cell cycle. Salvant
et al. (23) showed that following infection, no
immediate-early gene expression is detected until the cells have
entered the G1 phase of the cell cycle. Jordan et al.
(14) demonstrated that activation of HSV immediate-early
gene expression by the virion component VP16 requires the presence of a
G1-specific cyclin-dependent kinase. Therefore, cell
cycle-responsive immediate-early gene regulation appears to be
conserved among these different herpesvirus family members.
To date, all of the herpesvirus factors that have been shown to induce
cell growth arrest are virion components and/or immediate-early transcription factors (10). It appears then that
herpesviruses have evolved to replicate with a specific temporal
sequence of events that are intimately linked with the cell cycle.
Immediate-early gene expression is specifically induced by
G0- or G1-specific factors. Either virion
growth arrest proteins and/or the newly expressed immediate-early gene
products then elicit specific signaling to lock cells in the
appropriate stage of G0 or G1. Since
immediate-early gene expression also elicits parallel signaling of
viral DNA replication, this coupling of early viral events to the
G0 or G1 phase of the cell cycle may be
important to help prevent immediate-early genes from activating viral
DNA replication at an inappropriate point in the cell cycle.
The strong G2/M bias of Zta-positive cells following
exposure of Rael cells to 5-azacytidine is in striking contrast to the G0/G1 bias observed in the other lytic
cycle-inducing systems discussed here. A previous study has shown that
demethylation may play a role in the induction of Zta expression
(9). Therefore, it is possible that induction of Zta
following 5-azacytidine treatment of Rael cells occurs, at least in
part, through demethylation of its promoter during S phase. In this
case, Zta expression would be expected to be detected in late S phase
and/or in G2, which is consistent with the results reported
here. Interestingly, however, the Zta-positive cells appear to be
retained in G2/M phase (although there is clearly some
leakage, as evidenced at later times [Fig. 8 and 9]). We have
previously shown that Zta induces checkpoint functions that can affect
both the G1 and G2 checkpoints (5, 6). Furthermore, in some genetic backgrounds, Zta can
preferentially induce a G2 arrest (A. Rodriguez and E. K. Flemington, unpublished). Therefore, it is possible that 5-azacytidine
induces demethylation of the Zta promoter during S phase, activating
Zta expression, which in turn effects a G2 block.
Although demethylation of the Zta gene may play a role in activating
its expression, it is curious that there is an apparent delay of ca.
16 h before Zta can first be detected. If demethylation of Zta's
promoter fully explained the induction of Zta expression, then Zta
induction would be expected to occur immediately as the fractions of
the population that are in S phase progress to G2. It is
possible that hemimethylation is insufficient to induce Zta expression
and that cells must cycle twice before the Zta promoter is fully
demethylated and the Zta promoter becomes activated. On the other hand,
it is clear that 5-azacytidine induces a G1 arrest in a
portion of the population. It is possible that these arrested cells
secrete an autocrine factor that may cooperate with demethylation to
activate Zta gene expression. Further experiments will be required to
determine whether either of these scenarios are involved in the
regulation of Zta expression in this system.
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ACKNOWLEDGMENTS |
We thank Martin Allday, Gareth Inman, and George Bornkamm for
helpful discussions and for sharing unpublished information.
This work was supported by National Institutes of Health grant GM48045.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology, SL79, Tulane Cancer Center, 1430 Tulane Ave., New Orleans, LA 70112. Phone: (504) 988-1167. Fax: (504) 588-5516. E-mail: erik{at}flemingtonlab.com.
Present address: Centro de Biologia Molecular "Severo Ochoa,"
Facultad de Ciencias-Edif. Biologicas, Universidad Autonoma de Madrid,
28049 Cantoblanco, Madrid, Spain.
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Journal of Virology, May 2001, p. 4482-4489, Vol. 75, No. 10
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4482-4489.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
