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Journal of Virology, April 2009, p. 3436-3449, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.02349-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Transcriptional Coactivators Are Not Required for Herpes Simplex Virus Type 1 Immediate-Early Gene Expression In Vitro{triangledown}

Sebla B. Kutluay,1,2 Sarah L. DeVos,2,{dagger} Jennifer E. Klomp,2 and Steven J. Triezenberg1,2,3*

Graduate Program in Cell and Molecular Biology,1 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824,3 Van Andel Research Institute, Grand Rapids, Michigan 495032

Received 11 November 2008/ Accepted 21 January 2009


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ABSTRACT
 
Virion protein 16 (VP16) of herpes simplex virus type 1 (HSV-1) is a potent transcriptional activator of viral immediate-early (IE) genes. The VP16 activation domain can recruit various transcriptional coactivators to target gene promoters. However, the role of transcriptional coactivators in HSV-1 IE gene expression during lytic infection had not been fully defined. We showed previously that transcriptional coactivators such as the p300 and CBP histone acetyltransferases and the BRM and Brg-1 chromatin remodeling complexes are recruited to viral IE gene promoters in a manner dependent mostly on the presence of the activation domain of VP16. In this study, we tested the hypothesis that these transcriptional coactivators are required for viral IE gene expression during infection of cultured cells. The disrupted expression of the histone acetyltransferases p300, CBP, PCAF, and GCN5 or the BRM and Brg-1 chromatin remodeling complexes did not diminish IE gene expression. Furthermore, IE gene expression was not impaired in cell lines that lack functional p300, or BRM and Brg-1. We also tested whether these coactivators are required for the VP16-dependent induction of IE gene expression from transcriptionally inactive viral genomes associated with high levels of histones in cultured cells. We found that the disruption of coactivators also did not affect IE gene expression in this context. Thus, we conclude that the transcriptional coactivators that can be recruited by VP16 do not contribute significantly to IE gene expression during lytic infection or the induction of IE gene expression from nucleosomal templates in vitro.


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INTRODUCTION
 
Herpes simplex virus type 1 (HSV-1) is a large double-stranded DNA virus that establishes life-long latency in sensory neurons after an initial phase of lytic infection in epithelial cells. Viral gene expression during lytic infection is initiated by VP16, a tegument-associated transcriptional activator protein that stimulates the transcription of viral immediate-early (IE) genes (6). VP16 is recruited to viral IE gene promoters through cis-regulatory elements with a consensus sequence of 5'-TAATGARAT, in association with two host cell proteins, Oct-1 and HCF-1 (74). VP16, by its activation domain (AD), interacts with various general transcription factors and recruits the host RNA polymerase II (RNAP II) (12, 24, 29, 43, 75).

The packaging of eukaryotic DNA in the form of chromatin presents a significant impediment to the transcriptional machinery (42). This barrier can be overcome by the activator-dependent recruitment of coactivator protein complexes with either of two types of enzymatic activities. Some coactivators covalently modify histones by acetylation, methylation, phosphorylation, ubiquitinylation, sumoylation, ADP-ribosylation, or proline isomerization (30, 42). Some covalent histone modifications, such as the acetylation of lysine 9 and lysine 14 of histone H3 (H3K9/K14ac) or trimethylation of lysine 4 of histone H3 (H3K4me3), are marks of active transcription. In contrast, methylation of other lysine residues on histones is typically indicative of inactive transcription and heterochromatin formation (30). The second class of coactivators hydrolyzes ATP in the process of remodeling the position of nucleosomes along DNA or in removing nucleosomes from DNA (11, 59).

The interaction of transcriptional activators with coactivators has often been explored using a chimeric protein, Gal4-VP16 (58), comprising the DNA-binding domain of the Saccharomyces cerevisiae Gal4 protein and the AD of HSV-1 VP16. The VP16 AD can physically interact with and recruit transcriptional coactivators such as the histone acetyltransferases (HATs) p300 (KAT3B) and CBP (KAT3A) (3, 17, 25, 34, 67, 70), PCAF (KAT2B) (70) and GCN5 (KAT2A) (23, 38, 63, 67, 68), or the ATP-dependent chromatin remodeling enzymes BRM and Brg-1 (16, 46, 49, 50) to potentiate transcription from nucleosomal templates. However, the role of coactivators in the context of HSV-1 infection is not yet well defined, in part because of prior evidence that the HSV-1 genome is predominantly nonnucleosomal during lytic infection (40, 41, 47).

We and others have recently shown that histones, most often represented experimentally by histone H3, are present on the HSV-1 genome during lytic infection but at lower levels than cellular genes (20, 22, 28, 35, 52). Furthermore, active transcription marks such as H3K9/K14ac and H3K4me3 have been associated with viral genes during lytic infection (20, 22, 28, 35). We have also shown that at early times during lytic infection, the p300 and CBP HATs and the BRM and Brg-1 chromatin remodeling enzymes are recruited to viral IE gene promoters in a manner mostly dependent on the presence of VP16 AD (20). Similarly, the Set1 histone methyltransferase, which is recruited by HCF-1, was shown to contribute to optimal HSV-1 gene expression (22). These results suggest that during lytic infection, nucleosomes might be deposited on the viral genome, and yet the recruitment of transcriptional coactivators could result in the modification and removal of the histones from the viral genome similar to actively transcribed genes in the host cell genome (4, 19, 33, 56).

Based on this model, we have hypothesized that the transcriptional coactivators that are recruited by VP16 are required for IE gene expression during lytic infection. From this hypothesis, we predicted that disrupting the expression of a coactivator will diminish IE gene expression by allowing the formation of an inactive chromatin structure on the viral genome. Here, we show that, contrary to our hypothesis, disrupting the expression of various coactivators by RNA interference (RNAi) did not decrease IE gene expression in HSV-infected cells under most conditions tested. In parallel with these findings, IE gene expression was not impaired in SiHa cells, which do not express functional p300, or in SW13 and C33-A cells, neither of which express the BRM and Brg-1 remodeling enzymes. Moreover, the restoration of BRM and Brg-1 activity to SW13 or C33-A cells had no substantial effect on IE gene expression, indicating that neither BRM nor Brg-1 remodeling enzymes are essential for IE gene expression.

If not important for lytic infection, we then hypothesized that coactivators may be required during reactivation from latency, during which time the viral genomes are nucleosomal. We have not yet tested the requirement of coactivators during reactivation from latency in vivo; instead, we used in vitro conditions in which viral genomes are heavily occupied with nucleosomes in cultured cells. To this end, we employed a mutant virus strain (RP5) that lacks sequences encoding the AD of VP16. During RP5 lytic infection, IE gene expression is reduced dramatically (66, 76), and histones associate with the RP5 genomes at higher levels than the wild-type genomes (20). Moreover, p300 and CBP HATs or BRM and Brg-1 remodeling enzymes are not recruited efficiently to RP5 IE promoters (20). Although IE gene expression from the RP5 genome was induced significantly upon superinfection by HSV-2, none of the transcriptional coactivators were required for this induction. We conclude that the coactivators that are recruited by VP16 are not essential for IE gene expression during lytic infection in vitro regardless of the nucleosomal status of the viral genome.


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MATERIALS AND METHODS
 
Cell lines and viruses. HeLa (ATCC CCL-2), SW-13 (ATCC CCL-105), Vero (ATCC CCL-81), and telomerase-transformed human foreskin fibroblasts (HFFs) provided by Wade Bresnahan, were grown in Dulbecco's modified Eagle medium (Invitrogen) containing 110 mg/liter sodium pyruvate and 10% fetal bovine serum (Invitrogen). SiHa (ATCC HTB-35) and C33-A (ATCC HTB-31) cells were grown in minimum essential medium containing Earle's salts supplemented with nonessential amino acids, 1 mM sodium pyruvate, and 10% fetal bovine serum. HSV-1 strain RP5, which lacks sequences encoding the AD of VP16, and strain RP5R (RP5 rescue) were previously described (66). HSV-1 strains KOS, RP5R, and RP5 and HSV-2 strain G were prepared, and titers were determined using Vero cells. In some experiments, cycloheximide (100 µg/ml) was added to the medium for 30 min prior to and during infection to inhibit protein translation.

Plasmids and transfections. A p300 expression plasmid, pCI-FLAG-p300, was provided by Yoshihiro Nakatani (5). Expression plasmids pCG-BRM, pBJ-Brg-1, dnBRM, and dnBrg-1 were obtained from Bernard Weissman and David Reisman (1, 64). SiHa cells were transfected using jetPEI (Polyplus) transfection reagent according to the manufacturer's instructions. SW13 and C33-A cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

siRNAs and transfections. For each target coactivator, two siRNA duplexes were purchased from Qiagen, with the exception of CBP_1 (Dharmacon). The catalog numbers and sequences of siRNAs are given in Table 1. For siRNA transfections, 1.5 x 105 HHFs were plated per well in six-well cell culture plates 1 day prior to transfection. siRNA duplexes were transfected at a 10 nM (for single and double transfections) or 20 nM (for quadruple transfections) total concentration using Silentfect transfection reagent (Bio-Rad) according to the manufacturer's instructions, with the exception that the siRNA duplexes and the transfection reagent were diluted in Optimem reduced-serum medium (Invitrogen).


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  		TABLE 1. siRNA sequences and catalog numbers used in this study

Gene expression assays and Q-RT-PCRs. Total cellular RNA was isolated using Trizol reagent (Invitrogen). Total RNA was reverse transcribed using random primers in a commercial reverse transcription system (Promega). The cDNA was used as a template in quantitative real-time PCR (Q-RT-PCR) assays using SYBR green master mix (Roche) and an ABI 7500 real-time PCR system (Applied Biosystems). Gene expression was first normalized against 18S rRNA and then to appropriate controls by the 2–{Delta}{Delta}CT method. For chromatin immunoprecipitation assays, data were analyzed using the standard-curve method, which is explained in more detail below. Primer pairs used in this study are indicated in Table 2. Other primer pairs were previously described (20, 54). For statistical analysis of gene expression, four or more biological replicates of a given experiment were analyzed by Student's t test.


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  		TABLE 2. Oligonucleotides used as primers for Q-RT-PCRa

Immunoblotting. Total cell lysates were prepared with radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with protease inhibitors (10 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin and leupeptin). Twenty-five to fifty micrograms of the lysates was run on 6, 8, or 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Blots were blocked in 5% nonfat dry milk-TTBS (100 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Tween 20) overnight at 4°C under constant agitation. The blots were then incubated for 2 h with the primary antibodies diluted in TTBS supplemented with 1% bovine serum albumin (Invitrogen). Antibodies specific for p300 (SC-584), CBP (SC-369), BRM (SC-6450), Brg-1 (SC-10768), PCAF (SC-13124), and GCN5 (SC-6303) were obtained from Santa Cruz Biotechnology. Antibodies specific for CD44 (156-3C11) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (ab9484) were purchased from Cell Signaling Technology and Abcam, respectively. Blots were then incubated with the proper horseradish peroxidase-conjugated secondary antibodies and visualized using a chemiluminescence detection system (Pierce) and Chemi-Doc imaging system with Quantity One software (Bio-Rad).

Chromatin immunoprecipitation. Chromatin immunoprecipitation was performed as explained previously, with minor modifications (20). Briefly, confluent plates of HFF cells were infected with RP5 or RP5R strains of HSV-1 at an MOI of 0.025 PFU/cell and 5 PFU/cell, which corresponded to about 8 to 10 viral genomes per cell for each infection. At 6 h postinfection (p.i.), infections were stopped by the addition of formaldehyde to cell culture plates at a final concentration of 1%. Chromatin was isolated and sonicated using a Branson Digital Sonifier-450 to obtain 200-to 1,000-bp DNA fragments. Protein-DNA complexes were immunoprecipitated using 5 µg of antibodies against histone H3 (ab1791; Abcam). Protein-DNA complexes were collected by protein G-agarose beads (Invitrogen). After several washes, the protein-DNA complexes were eluted and reverse cross-linked overnight at 65°C in the presence of 200 mM NaCl and 10 µg RNase A. Samples were then precipitated with ethanol, digested with proteinase K (Roche) at 42°C for 2 h, and purified with Qiagen spin columns using the gel extraction protocol. The presence of viral and cellular DNA fragments in the immunoprecipitated material was analyzed by quantitative real-time PCR using SYBR green master mix (Roche) and an ABI 7500 real-time PCR system (Applied Biosystems). A standard curve using serial threefold dilutions of input samples (1, 0.3, 0.1, and 0.04%) was produced to quantitate the signals from immunoprecipitation samples. Background signals, obtained from immunoprecipitation reactions performed in the absence of antibodies (no-antibody control), were subtracted from signals obtained from immunoprecipitation samples [referred to as "% input (IP-noab)"]. When necessary, data were further normalized against the cellular control U3 snRNA promoter by dividing the "% input (IP-noab)" value for the viral DNA by that of the cellular DNA to account for the differences in immunoprecipitation efficiencies.


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RESULTS
 
RNAi of p300 and CBP HATs does not diminish HSV-1 IE gene expression. The related HAT enzymes p300 and CBP can potentiate VP16 AD-dependent transcriptional activation from reconstituted nucleosome arrays in vitro or from reporter plasmids in vivo (3, 17, 67, 70). We previously showed that both p300 and CBP are recruited to IE gene promoters in a manner that is dependent mostly on the presence of VP16 AD (20). Others also indicated that p300 and CBP are recruited to PML bodies that become viral replication compartments later in infection (37, 44, 45). However, a direct role for p300/CBP in viral gene expression has not yet been established. The present study was undertaken to address this gap in our understanding.

We have previously shown that disrupting the expression of p300 by plasmid-based RNAi in HeLa cells did not affect IE gene expression (35). However, this finding was complicated by several considerations. First, p300 and CBP in some contexts have been shown to be redundant (26, 69), and therefore, knocking down p300 itself may not have been sufficient to affect IE gene expression. Second, RNAi is not 100% efficient, and thus, the residual levels of p300 might have been sufficient for IE gene expression. Third, analysis of coactivators in HeLa cells might be inherently flawed due to the presence in HeLa cells of the papillomaviral proteins E6 and E7, which affect the activities of p300 and CBP (55, 79).

To overcome these potential problems, we disrupted the expression of p300 and CBP, both separately and in combination, by multiple small interfering RNA (siRNA) duplexes in telomerase-transformed HFFs. Steady-state levels of p300 and CBP proteins (Fig. 1A) and mRNA (Fig. 1B) were significantly and specifically reduced by siRNA duplexes designed to target these two related proteins. One siRNA duplex targeting p300 (p300_1) reduced p300 protein expression more than p300 mRNA expression, which likely reflects a block in mRNA translation rather than mRNA degradation.


Figure 1
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  		FIG. 1. Disruption of p300 and CBP expression by RNAi does not decrease HSV-1 IE gene expression. HFFs were transfected with siRNA duplexes targeting p300, CBP, or a negative control nontargeting siRNA duplex. (A) Immunoblot showing p300, CBP, and GAPDH protein levels 48 h after siRNA transfection. The arrow indicates the CBP-specific band. (B) Q-RT-PCR analysis of p300 and CBP expression in siRNA-transfected and KOS-infected HFFs. Values for each target are represented relative to the negative control siRNA signal. Histograms represent the averages of data from six independent experiments, and error bars represent the standard deviations. (C) siRNA-transfected HFFs were infected with HSV-1 KOS at an MOI of 10 PFU/cell. Expression of viral IE genes (ICP0, ICP4, and ICP27) at 2.25 h p.i. was analyzed by Q-RT-PCR. (D) siRNA-transfected HFFs were infected with HSV-1 at an MOI of 0.1 PFU/cell. IE gene expression at 4 h p.i. was analyzed by Q-RT-PCR. (E) siRNA-transfected HFFs were pretreated with 100 µg/ml cycloheximide for 30 min and then infected with HSV-1 at an MOI of 0.1 PFU/cell in the presence of 100 µg/ml cycloheximide for 4 h. IE gene expression was analyzed by Q-RT-PCR. Data in C, D, and E represent the averages of data from two independent experiments, each done with biological duplicates. Error bars represent standard deviations based on these four samples. Mean values that differ significantly from those obtained from cells transfected with negative control siRNA are indicated by * for P < 0.01 or by # for 0.01 < P < 0.05 as determined by Student's t test.

We then tested whether IE gene expression was reduced by the disruption of p300 and CBP in HFFs. To address this, HFFs were transfected with siRNA duplexes as described above and were then infected with wild-type HSV-1 at a multiplicity of infection (MOI) of 10 PFU per cell. At 2.25 h p.i., when IE gene expression is robust, total RNA was isolated and further processed for analysis of IE gene expression. To our surprise, the disruption of neither p300 nor CBP resulted in a reduced level of expression of ICP0, ICP4, or ICP27 mRNA (Fig. 1C). Moreover, even the simultaneous knockdown of p300 and CBP had no deleterious effect on viral IE gene expression. One of the p300-specific siRNA duplexes (p300_2), either alone or in combination with the CBP_2 duplex, resulted in a statistically significant increase in IE gene expression, quite the contrary of the expected outcome. We suspect that this increase represents an off-target effect of that specific siRNA, as the other duplex that also targets p300 (p300_1) did not show a similar effect.

The initial assays shown in Fig. 1C were conducted using relatively high MOIs (10 PFU/cell). We considered whether a requirement for p300 and CBP might be more evident during low-multiplicity infections, in which the viral genome might be more prone to transcriptional repression by the deposition of host histones. HFF cells transfected with the various siRNA duplexes were infected at a low MOI (0.1 PFU/cell), and RNA harvested at 4 h p.i. was analyzed for IE gene expression using Q-RT-PCR (Fig. 1D). The results (Fig. 1D) are comparable to those for the high-MOI infections; siRNAs targeting p300 or CBP (or both together) have no deleterious effect on viral IE gene expression. In fact, levels of ICP0 and ICP27 but not ICP4 expression showed a modest but statistically significant increase when p300 and CBP are knocked down either separately or together. These results suggest that p300 and CBP are not required for IE gene expression at a low MOI, and if anything, they may act to repress IE gene transcription.

The IE gene products ICP4 and ICP0 themselves have activities that regulate IE gene transcription; for example, ICP0 might bypass the requirement for coactivators by disrupting the REST/CoREST/HDAC repressor complex (13, 14). To prevent any feedback on IE gene expression by the IE proteins themselves, parallel experiments were conducted in the presence of cycloheximide, a translation inhibitor. The results (Fig. 1E) again show that siRNAs targeting p300 or CBP have no deleterious effect on viral IE gene expression. The disruption of p300 by the p300_2 duplex, either alone or in combination with CBP_2, caused a statistically significant increase in levels of expression of all IE genes during low-multiplicity infections (Fig. 1D and E), an observation that contradicts our original hypothesis. This increase is likely due to an off-target effect of the p300_2 siRNA duplex, since the other p300 siRNA did not have a similar effect. We conclude that the HATs p300 and CBP are neither required nor redundant for the activation of IE gene expression by VP16.

IE gene expression is not impaired in SiHa cells but is augmented by the expression of wild-type p300. Although p300 was knocked down efficiently by siRNAs in HFFs, we were concerned that the residual expression of p300 might still be sufficient to enable IE gene expression. To address this, we analyzed IE gene expression in SiHa cervical carcinoma cells, which express a mutated form of p300 that lacks the bromodomain (53). Since both SiHa and HeLa cells are derived from cervical carcinomas and are transformed by human papillomaviruses, we reasoned that comparing IE gene expression in these two cell lines would be a legitimate approach to test whether p300 is required for the transcription of IE genes. SiHa and HeLa cells were infected with KOS at 1 PFU/cell, and IE gene expression at 2 h p.i. was analyzed by Q-RT-PCR. Contrary to our hypothesis, IE gene expression in SiHa cells was not significantly different than that in HeLa cells (Fig. 2A). We also tested whether supplementing SiHa cells with fully functional p300 might further enhance viral IE gene expression. To that end, SiHa cells were transfected with a wild-type p300 expression plasmid or an empty plasmid. The overexpression of wild-type p300 in SiHa cells (Fig. 2B) resulted in increases in ICP0, ICP4, and ICP27 expression levels that were modest but statistically significant (Fig. 2C), suggesting that although p300 is not required, it may potentiate the transcription of viral IE genes in SiHa cells.


Figure 2
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  		FIG. 2. HSV-1 IE gene expression is not impaired in SiHa cells but is augmented in the presence of wild-type p300. (A) HeLa and SiHa cells were infected with HSV-1 strain KOS at an MOI of 1 PFU/cell. IE gene (ICP0, ICP4, and ICP27) and p300 mRNA levels at 2 h p.i. were analyzed by Q-RT-PCR. Values for each gene tested with SiHa cells are represented relative to those with HeLa cells. Error bars show the ranges between the averages of two independent experiments. (B) SiHa cells were transfected with 2.5 µg of plasmid pCI (empty) or pCI-p300. p300 and GAPDH expressions were analyzed 24 h after transfection by immunoblotting. (C) SiHa cells were transfected as described for B and were infected with HSV-1 KOS at an MOI of 5 PFU/cell. IE gene expression (ICP0, ICP4, and ICP27) at 2 h p.i. was analyzed by Q-RT-PCR. Values for each viral gene tested in pCI-p300-transfected cells are represented relative to cells transfected with empty plasmid (pCI). Error bars indicate standard deviations (n = 4). Mean values that vary significantly (P < 0.01 by Student's t test) from those obtained from cells transfected with vector plasmid are indicated (*).

Disruption of PCAF and GCN5 HATs does not affect HSV-1 IE gene expression. In addition to p300 and CBP, other HATs such as PCAF and GCN5 are known to interact with the AD of VP16 (68, 70). We therefore considered whether PCAF and GCN5 might be preferentially required for IE gene expression. To address this, IE gene expression was analyzed in HFFs in which the expression of PCAF and GCN5 was diminished either separately or together by siRNA duplexes. The siRNAs targeting PCAF and GCN5 in HFFs were both effective and specific for their respective targets, as indicated by immunoblotting (Fig. 3A) and Q-RT-PCR (Fig. 3B). We then infected these cells with HSV-1 at a high MOI (10 PFU/cell) and analyzed IE gene expression at 2.25 h p.i. As we observed for p300 and CBP, IE gene expression was not affected significantly under most circumstances when PCAF and GCN5 (separately or in combination) were knocked down (Fig. 3C). Transfection of two siRNAs (PCAF_1 and GCN5_2) caused a slight but statistically significant decrease in IE gene expression. However, since comparable effects were not exhibited by the PCAF_2 and GCN5_1 duplexes, which block target protein expression to levels similar to those of the PCAF_1 and GCN5_2 duplexes, we cannot attribute these modest changes in IE gene expression to the effects on the disruption of PCAF or GCN5. Parallel experiments were performed at a low MOI (0.1 PFU/cell) to see whether a requirement for PCAF and GCN5 might be more apparent under conditions of a lighter viral genome load. In most cases, IE gene expression did not change significantly when levels of PCAF and GCN5 expression were reduced (Fig. 3D). Although the GCN5_1 duplex reduced IE gene expression, the GCN5_2 duplex caused the opposite effect. Therefore, although these siRNAs alter IE gene expression, we do not think that these modest effects on IE gene expression are biologically meaningful. To rule out the possibility that IE proteins themselves were masking a requirement for coactivators in the activity of VP16 on IE gene expression, we repeated the low-MOI experiments in the presence of cycloheximide to block IE protein expression. Although the decrease in levels of ICP0 and ICP4 expression when GCN5 was knocked down (with either of two siRNA duplexes) was statistically significant, in general, the expression of IE genes was not impaired when PCAF and GCN5 were knocked down either separately or in combination (Fig. 3E). Collectively, these results suggest that although VP16 can recruit PCAF and GCN5 in artificial contexts, PCAF and GCN5 are neither required substantially nor redundant for IE gene transcription during lytic infection.


Figure 3
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  		FIG. 3. RNAi of PCAF and GCN5 does not decrease HSV-1 IE gene expression. HFFs were transfected with siRNA duplexes targeting PCAF, GCN5, or a negative control nontargeting siRNA duplex. (A) Western blots showing levels of PCAF, GCN5, and GAPDH proteins 48 h after siRNA transfection. (B) Q-RT-PCR analysis of PCAF and GCN5 expression in siRNA-transfected and KOS-infected HFFs. Data represent the averages of data from six independent experiments, and error bars represent the standard deviations. (C) siRNA-transfected HFFs were infected with HSV-1 at an MOI of 10 PFU/cell. Total RNA was isolated at 2.25 h p.i., and IE gene mRNA levels (ICP0, ICP4, and ICP27) were analyzed by Q-RT-PCR. (D) siRNA-transfected HFFs were infected with HSV-1 at an MOI of 0.1 PFU/cell. IE gene expression at 4 h p.i. was analyzed by Q-RT-PCR. (E) siRNA-transfected HFFs were pretreated with 100 µg/ml cycloheximide for 30 min and then infected with HSV-1 at an MOI of 0.1 PFU/cell in the presence of cycloheximide. IE gene expression at 4 h p.i. was analyzed by Q-RT-PCR. C to E present the averages of four biological replicates, and error bars represent standard deviations. (F) HFFs were transfected with siRNA duplexes targeting p300, CBP, PCAF, GCN5, or a negative control nontargeting siRNA duplex and infected with HSV-1 at an MOI of 10 or 0.1 PFU/cell. IE gene expression levels at 2.25 and 4 h p.i. for high and low MOIs, respectively, were analyzed by Q-RT-PCR. Data shown are from a representative experiment done with biological triplicates; error bars indicate the standard deviations. Experimental samples whose mean values differ significantly from those obtained from cells transfected with negative control siRNA are indicated by * for P < 0.01 or by # for 0.01 < P < 0.05 as determined by Student's t test.

To address whether different classes of HATs are redundant for IE gene expression, we then simultaneously disrupted the expression of p300, CBP, PCAF, and GCN5 HATs in HFFs and infected these cells with HSV-1 at high (10 PFU/cell) or low (0.1 PFU/cell) MOIs. As indicated in Fig. 3F, regardless of the MOI, the disruption of all four HATs did not reduce viral IE gene expression; if anything, we observed a slight but significant increase in levels of ICP0 and ICP27 expression. These results suggest that p300, CBP, PCAF, and GCN5 HATs are not redundant for IE gene expression during lytic infection in vitro.

RNAi of BRM and Brg-1 chromatin remodeling complexes does not affect IE gene expression. Several lines of evidence indicate that ATP-dependent chromatin remodeling complexes can be recruited by the AD of VP16 to nucleosomal templates, leading to the disruption of nucleosomes and transcriptional activation (46, 49, 50, 72, 78). The VP16 AD can also enhance nucleosome eviction by the SWI/SNF remodeling complex from mononucleosomal templates in vitro (16). Therefore, we hypothesized that BRM and Brg-1 chromatin remodeling complexes, the mammalian homologues of the yeast SWI/SNF complex (72), might remove the nucleosomes from the viral genome and enable active transcription.

To address this, as in above-described sections, we analyzed IE gene expression in HFFs in which the expression of BRM and Brg-1 remodeling enzymes was disrupted by RNAi, with the expectation that IE gene expression would diminish in the absence of BRM and Brg-1. Immunoblotting (Fig. 4A) and Q-RT-PCR (Fig. 4B) results indicate that BRM and Brg-1 were knocked down very efficiently and specifically by both siRNA duplexes against each target. However, the disruption of BRM and Brg-1 expression by most siRNA duplexes (with the exception of Brm_1) did not reduce IE gene expression at a high MOI (10 PFU/cell) during lytic infection (Fig. 4C). On the contrary, IE gene expression was increased in the presence of some siRNAs, most notably, Brg-1_2. In parallel experiments performed at a low MOI (0.1 PFU/cell), the transcription of some IE genes during lytic infection was affected by one but not by both of the siRNA duplexes targeting either BRM or Brg-1 (Fig. 4D). Therefore, we conclude that reduced levels of the BRM or Brg-1 remodeling enzymes do not affect IE gene transcription substantially. Moreover, similar results were also obtained when the viral infection was performed in the presence of cycloheximide to prevent the interference of IE proteins in IE gene transcription (Fig. 4E). Interestingly, under all conditions tested, levels of expression of most IE genes increased significantly when Brm and Brg-1 were knocked down together (Fig. 4C, D, and E), suggesting that Brm and Brg-1 might be acting redundantly in a manner contrary to our original hypothesis, i.e., to inhibit rather than to support IE gene expression. Although primarily associated with transcriptional activation, Brm and Brg-1 have been shown to potentiate transcriptional repression by the Rb tumor suppressor protein (15).


Figure 4
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  		FIG. 4. Disruption of BRM and Brg-1 expression does not decrease HSV-1 IE gene expression. HFFs were transfected with the indicated siRNA duplexes targeting BRM, Brg-1, or a negative control nontargeting siRNA duplex. (A) Western blot showing BRM, Brg-1, and GAPDH expression in HFFs 48 h after siRNA transfection. (B) Q-RT-PCR analysis of BRM and Brg-1 in HFFs after siRNA transfection and KOS infection. Data represent the averages of data from six independent experiments. Error bars represent the standard deviations. (C) After siRNA transfection, HFFs were infected with HSV-1 at an MOI of 10 PFU/cell. IE gene expression at 2.25 h p.i. was analyzed by Q-RT-PCR. (D) siRNA-transfected HFFs were infected with HSV-1 at an MOI of 0.1 PFU/cell. IE gene expression at 4 h p.i. was analyzed by Q-RT-PCR. (E) After siRNA transfection, HFFs were pretreated with 100 µg/ml cycloheximide for 30 min and then infected with HSV-1 at an MOI of 0.1 PFU/cell in the presence of 100 µg/ml cycloheximide. Total RNA was isolated at 4 h p.i., and IE gene expression was analyzed by Q-RT-PCR. C to E present the means of data from four biological replicates; error bars represent standard deviations. Mean values that differ significantly from those obtained from cells transfected with negative control siRNA are indicated by * for P < 0.01 or by # for 0.01 < P < 0.05 as determined by Student's t test.

IE gene expression in cell lines with defective BRM and Brg-1 expression. As a strategy complementary to RNAi, we also analyzed IE gene expression in cell lines that do not express BRM and Brg-1 enzymes (57, 73). The immunoblot shown in Fig. 5A confirms that the levels of BRM and Brg-1 proteins are dramatically reduced in SW13 (adrenal carcinoma) and C33-A (cervical carcinoma) cells compared with HeLa cells.


Figure 5
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  		FIG. 5. HSV-1 IE gene expression in SW13 and C33-A cells that do not express BRM and Brg-1 remodeling enzymes. (A) BRM, Brg-1, and GAPDH expression in HeLa, SW13, and C33-A cells was analyzed by immunoblotting. (B and C) HeLa, SW13, and C33-A cells were infected in parallel at an MOI of 5 PFU/cell (B) or 0.1 PFU/cell (C). IE gene expression (ICP0, ICP4, and ICP27) at 2 h p.i. was analyzed by Q-RT-PCR. IE gene expression in SW13 and C33-A cells is represented with respect to that in HeLa cells. The graph shows the average of data from two independent experiments each done in biological quadruplicate (B) or triplicate (C). Error bars represent the ranges between the averages of these experiments. Mean values that vary significantly (P < 0.01 by Student's t test) from those obtained from HeLa cells in both of the experiments presented in B and C are indicated (*). (D) SW13 cells were transfected with 4 µg of an empty plasmid or with expression plasmids encoding BRM, Brg-1, dominant negative BRM (dnBRM), or dominant negative Brg-1 (dnBrg-1) together with 0.5 µg of puromycin selection plasmid. Twenty-four hours posttransfection, medium was replaced by puromycin selection medium (2.5 µg/ml puromycin). After 2 days of puromycin selection, total protein was isolated and analyzed by immunoblotting against BRM, Brg-1, and GAPDH. (E and F) SW13 cells were transfected with the indicated plasmids as described above (D) and infected with HSV-1 KOS at an MOI of 0.1 PFU/cell. At 2 h p.i., total RNA was isolated, and CD44 (E) or IE gene expression (F) was analyzed by Q-RT-PCR. (G) Immunoblot showing BRM, Brg-1, CD44, and GAPDH expression in C33-A cells transfected with the indicated plasmids as described above (D). (H and I) C33-A cells were transfected with the indicated plasmids as described above (D) and infected with HSV-1 KOS at an MOI of 0.1 PFU/cell. At 2 h p.i., total RNA was isolated, and levels of CD44 (H) and IE (I) gene expression were analyzed by Q-RT-PCR. Data for E, F, H, and I are derived from a representative experiment done with biological triplicates; error bars represent standard deviations. Mean values that vary significantly (P < 0.01 by Student's t test) from those obtained from cells transfected with vector plasmid are indicated (*).

We then asked whether IE gene expression was impaired in SW13 and C33-A cells. Parallel cultures of HeLa, SW13, and C33-A cells were infected with HSV-1 at high (5 PFU/cell) or low (0.1 PFU/cell) MOIs. Q-RT-PCR assays of viral IE gene expression revealed no defect in SW13 cells and about a twofold increase in C33-A cells in the high-multiplicity infections (Fig. 5B). At a low MOI (0.1 PFU/cell), IE gene expression was about 60% lower in SW13 cells but twofold higher in C33-A cells (Fig. 5C). Given that both cell lines are defective for BRM and Brg-1, we cannot attribute the deficit in SW13 cells at low MOIs to a requirement for these coactivators.

To further test for potential contributions by BRM and Brg-1, we transfected SW13 or C33-A cells with plasmids expressing either wild-type BRM or Brg-1 or dominant negative forms that lack ATPase activity (Fig. 5D and G). The endogenous gene encoding the cell surface marker CD44 served as a positive control, since CD44 is known to be regulated by BRM and Brg-1 in these cells (64, 65). As expected, CD44 mRNA expression was induced in both SW13 and C33-A cells upon the expression of BRM and Brg-1 but not the dominant negative BRM and Brg-1 (Fig. 5E and H), indicating that the BRM and Brg-1 proteins ectopically expressed in these cell lines are functional. When these cells were subsequently infected with HSV-1, IE gene expression in SW13 cells that express wild-type BRM and Brg-1 was not significantly different (P > 0.05 by Student's t test) from that in cells that were transfected with an empty plasmid or with plasmids encoding the dominant negative forms of BRM or Brg-1 (Fig. 5F). Curiously, IE gene expression in C33-A cells expressing wild-type BRM or Brg-1 was approximately 60% lower than that in parallel cells transfected with empty vector (Fig. 5I). However, this reduction seems to be independent of the catalytic activity of BRM and Brg-1, since the expression of the dominant negative forms of BRM and Brg-1 also reduced IE gene expression to similar levels (Fig. 5I). This suggests that BRM and Brg-1 do not repress IE gene expression, as suggested above by RNAi assays (Fig. 4). In other words, if BRM and Brg-1 were inhibitory for IE gene expression, restoring BRM and Brg-1 expression in both SW13 and C33-A cells would decrease IE gene expression. These results together with the RNAi assays described above lead us to conclude that the chromatin remodeling enzymes BRM and Brg-1 are neither required nor redundant for IE gene expression during lytic infection.

Coactivators are not required for VP16-mediated induction of IE gene expression from nucleosomal viral genomes in vitro. One potential reason why transcriptional coactivators are not required for IE gene transcription during lytic infection is that the viral genomes remain depleted of histones by some undefined mechanism that may bypass the need for coactivators. To explore this question, we designed an experiment in which histones can be more abundantly deposited on viral genomes prior to the introduction of transcriptionally active VP16. We have shown previously that IE gene expression is dramatically reduced during lytic infection by RP5, a mutant virus that lacks sequences encoding the AD of VP16 (66, 76). In addition, recruitment of the p300 and CBP HATs or the BRM and Brg-1 chromatin remodeling enzymes to most IE promoters is also impaired in RP5 infections (20). Furthermore, histones (represented by histone H3) associate with RP5 genomes to a greater extent than with wild-type genomes (20). Therefore, to some extent, RP5 infection resembles quiescent or latent infections with respect to defects in IE gene expression and increased histone occupancy on the viral genome.

We first asked whether IE genes in the RP5 genome could be activated by superinfection with HSV-2, which encodes a VP16 protein very similar to that of HSV-1 (9) and which can induce reactivation from quiescence in other contexts (8). HFFs were infected with RP5 at MOIs ranging from 0.0005 PFU/cell to 0.05 PFU/cell, which correspond to approximately 0.5 to 1 viral genome per cell to 50 to 100 viral genomes per cell, respectively (data not shown). At 6 h p.i., histone deposition on RP5 genomes was dramatically higher than that on wild-type genomes (Fig. 6A). The RP5-infected cells were then superinfected with HSV-2 strain G at MOIs ranging from 0.1 PFU/cell to 10 PFU/cell. Two hours after initiating the HSV-2 infection, we assayed levels of HSV-1-specific IE gene expression by Q-RT-PCR. As expected, superinfection of RP5-infected cells with HSV-2 activated the expression of ICP4 (Fig. 6B) and ICP27 (Fig. 6C) in a dose-dependent manner with respect to the HSV-2 MOI. These results indicate that the defect in IE gene expression in RP5 infections can be overcome effectively by providing VP16 in trans by HSV-2 superinfection. In subsequent assays, we have performed RP5 and HSV-2 infections at MOIs of 0.005 PFU/cell and 10 PFU/cell, respectively, to obtain robust IE transcription.


Figure 6
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  		FIG. 6. HSV-2 superinfection induces IE gene expression in RP5-infected cells. (A) HFFs were infected with HSV-1 strain RP5 or RP5R at an MOI of 0.025 PFU/cell or 5 PFU/cell, respectively. At 6 h p.i., chromatin immunoprecipitation (ChIP) was performed to assay for the presence of histone H3 on the ICP0 promoter, ICP27 promoter, ICP27 open reading frame (orf), tk promoter, and gC promoter. (B and C) HFFs were infected with HSV-1 RP5 at an MOI of 0.05, 0.005, or 0.0005 PFU/cell. At 6 h after RP5 infections, superinfection with HSV-2 was performed at an MOI of 0.1, 1, or 10 PFU/cell. Levels of ICP4 (B) and ICP27 (C) expression at 2 h after HSV-2 superinfection were analyzed by Q-RT-PCR.

To address whether coactivators are required for the HSV-2-mediated expression of IE genes from the quiescent RP5 genomes, we disrupted the expression of each coactivator in HFFs by siRNAs, infected these cells with RP5, and then superinfected these cells with HSV-2. Contrary to our hypothesis, IE gene expression from the RP5 genomes was effectively stimulated by HSV-2 superinfection even in the presence of siRNAs targeting any of the coactivators (Fig. 7). If anything, the siRNAs resulted in a modest increase, rather than a decrease, in IE gene expression. These results suggest that coactivators are not required for the VP16-mediated induction of IE gene transcription from viral DNA that is abundantly associated with histones.


Figure 7
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  		FIG. 7. Coactivators are not required for HSV-2-dependent induction of IE gene expression from RP5 genomes. HFFs were transfected with the indicated siRNA duplexes targeting p300 and CBP (A), PCAF and GCN5 (B), BRM and Brg-1 (C), or a negative control nontargeting siRNA duplex. Forty-eight hours after transfection, HFFs were infected with RP5 at an MOI of 0.005 PFU/cell. At 6 h after RP5 infection, HFFs were superinfected with HSV-2 at an MOI of 10 PFU/cell. HSV-1 IE gene expression at 2 h after HSV-2 infection was analyzed by Q-RT-PCR. The graph displays data for the means of four biological replicates, and error bars denote standard deviations. Mean values that differ significantly from those obtained from cells transfected with negative control siRNA are indicated by an * for P < 0.01 or by # for 0.01 < P < 0.05 as determined by Student's t test.


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DISCUSSION
 
Although a number of prior reports described evidence that the HSV-1 genome remains nonnucleosomal during lytic infection (40, 41, 47), several groups have recently shown that the viral genome is not exclusively histone free and that acetylated and methylated histones are present on the viral genome during lytic infection albeit at lower levels than on cellular genes (20, 22, 28, 35, 52). In contrast, during latency, the viral genome is packaged in a manner more nearly resembling that of host cell chromatin (10). Histones on the latent HSV-1 genomes carry posttranslational modifications typical of heterochromatin, with the exception of the actively transcribed latency-associated transcript gene (32, 71). During reactivation from latency or quiescence, the histones associated with IE gene promoters and other regions of the genome become acetylated, a hallmark of transcriptionally active chromatin (8, 51). These observations all indicate that the regulation of chromatin might play a crucial and distinct role in different stages of HSV-1 infection.

We do not yet understand how the viral genome manages to stay predominantly histone free during lytic infection. Given that VP16 AD interacts with a number of transcriptional coactivators in artificial conditions and that some of these coactivators are recruited to IE gene promoters during lytic infection (20), we hypothesized that these coactivators are involved in establishing a transcriptionally active chromatin state on the viral genome and enable IE gene expression. To this end, we tested whether disrupting the expression of these coactivators would decrease IE gene expression. Reducing the expression levels of p300 and CBP HATs, singly or in combination, using siRNAs had no discernible effect on IE gene expression at different MOIs (Fig. 1), suggesting that neither p300 nor CBP is required for IE gene expression. The modest increase in levels of IE gene expression observed following the transfection of one of the p300 duplexes, p300_2, is attributed to an off-target effect of this siRNA duplex, since the other siRNA was just as effective in diminishing p300 protein levels but had no effect on viral IE mRNA levels. An alternative explanation that we cannot exclude is that the diminished level of expression of p300 seen in the presence of siRNA p300_2 might suppress the host innate immune defense and hence enhance IE gene expression as suggested by others previously (45). Disruption of other HATs, PCAF and GCN5, by RNAi also had no significant effect on IE gene expression under most conditions tested in vitro (Fig. 3). Together, these results lead us to reject the hypothesis that histone acetylation is required for IE gene expression during lytic infection in vitro in HFFs.

Given that the AD of VP16 can stimulate nucleosome eviction by the yeast SWI/SNF remodeling complex (16), we hypothesized that BRM and Brg-1, the mammalian homologues of SWI/SNF, may be required for removing the nucleosomes from the viral genome. However, the disruption of BRM and Brg-1, separately or together, did not reduce IE gene expression (Fig. 4). Although RNAi of BRM and Brg-1 together increased the levels of expression of most IE genes under some conditions, these results were not supported by our findings with SW13 and C33-A cells. IE gene expression was not impaired in SW13 and C33-A cells, which express neither BRM nor Brg-1 (Fig. 5B and C), and restoring BRM and Brg-1 expression in these cell lines did not increase IE gene expression (Fig. 5F and I). Collectively, these results indicate that BRM and Brg-1 do not have a substantial role in supporting the VP16-dependent activation of viral IE gene expression during lytic infection in vitro.

The results of these experiments suggest that the transcriptional coactivators tested here are not required for IE gene expression during lytic infection, in contrast with our initial hypothesis. We recognize that cell culture models of HSV-1 infection are not necessarily representative of in vivo infections in epithelial or neuronal cells. Therefore, future studies will be necessary to test whether coactivators are important in lytic infection in vivo.

One potential explanation for the lack of an effect is redundancy or compensation among coactivators such that the activity lost by the disruption of one coactivator is taken up by another coactivator. We have addressed this in part by analyzing IE gene expression in HFFs where two or more coactivators were simultaneously disrupted. However, even when as many as four coactivators were targeted by siRNAs, viral IE gene did not decrease (Fig. 3F). Nonetheless, we cannot fully exclude the possibility that other HATs or remodeling complexes are compensating for those disrupted in our experiments.

We used immunoblots of the targeted proteins as an indication of the effectiveness of the siRNAs employed in these experiments. Although the targeted protein levels were substantially diminished (to levels 20% or less relative to levels of control cells), the residual protein may be sufficient for the biological activities that we have tested. Unfortunately, in most cases, no suitable positive control genes have been identified; that is, genes that are known to be direct targets of a given coactivator in HFFs. The exception may be the CD44 gene in SW13 and C33-A cells, which clearly responded to the presence of wild-type but not mutant forms of Brg-1 or BRM. In addition, as suggested in a recent study (21), we observed that the disruption of p300 and CBP HATs together led to a substantial decrease in H3K18ac levels (data not shown), suggesting that our RNAi of CBP and p300 expression was effective. To gain confidence in the outcomes of the siRNA experiments, we also took the complementary approach of testing IE gene expression in mutant cell lines that lack the particular coactivator. The consistency of the results from these two approaches strongly supports our conclusion that these transcriptional coactivators are not intimately involved in HSV-1 IE gene expression.

Although the coactivators tested in this study are not required for IE gene expression, other coactivators might be required for the modification of chromatin structure on the viral genome. By employing similar assays, others have shown that the Set1 histone methyltransferase can be recruited by HCF-1 and can contribute to viral gene expression during lytic infection (22). Although the disruption of Set1 expression did not significantly affect IE gene expression at early times in infection, at later stages viral gene expression and viral replication were reduced modestly. Similarly, during lytic infection by varicella zoster virus (like HSV-1, a member of the alphaherpesvirus family), Set1 recruitment to the IE62 promoter was correlated with high levels of H3K4me3 (48). However, whether Set1 is required for IE62 expression has not been explicitly tested. Therefore, further studies are necessary to ask whether Set1 or other coactivators are important for IE gene transcription.

Another possibility is that VP16 recruits transcriptional coactivators not for IE gene expression during lytic infection but during reactivation from latency, where the viral genome is nucleosomal (10). We have addressed this in part by testing whether coactivators are required during the activation of IE gene expression from histone-laden viral genomes in vitro. HSV-1 strain RP5 lacks the VP16 AD, and the density of histone H3 on the RP5 genome approaches that of cellular genes at later times in infection (Fig. 6A). Superinfection by HSV-2 of RP5-infected cells resulted in a substantial IE expression from the RP5 template (Fig. 6). However, knocking down the expression of various coactivators had little or no effect on RP5 IE gene expression (Fig. 7), suggesting that the VP16-mediated reactivation of IE gene expression does not require the coactivators tested in this study. We recognize that this in vitro system is not a genuine representation of latent infection and that some experiments reported previously by others suggested that VP16 may not be required for reactivation (62). Nonetheless, these experiments do address the role of coactivators for VP16-mediated transcription from a predominantly nucleosomal template. Future studies will be necessary to more directly establish whether coactivators are required in other quiescent infection models or, most importantly, during reactivation from latency in vivo.

A final possibility is that these coactivators may not be required at all for HSV-1 gene expression; their apparent presence at IE gene promoters may simply reflect their association with larger complexes of the transcription machinery. Recruitment of a particular transcription factor does not always correspond to a functional requirement for that factor. For instance, although estrogen receptor {alpha} can bind to a large number of cellular promoters, only about 10% of those genes are actually regulated by estrogen (36).

Given that the transcriptional coactivators tested in this study are not required for IE gene transcription, the mechanism by which VP16 stimulates IE gene transcription is still not clear. Although current models suggest that histones are first deposited on the viral genome at early times in infection and then removed, no evidence indicates that histone deposition precedes IE gene transcription. An alternative to the model that histones are deposited, modified, and then removed is a model in which the deposition of histones on the viral genome is prevented by an as-yet-undefined mechanism. If this is the case, then coactivators would not be required for the transcription of IE genes. Alternatively, other mechanisms, such as the recruitment of Set1 histone methyltransferase by HCF-1 (22, 48) or the disruption of the REST/CoREST/HDAC repressor complex by ICP0 (13, 14), may be the major determinant for histone depletion on the viral genome. In support of ICP0 having a role in chromatin dynamics during HSV-1 infections, a recent study indicated that the absence of ICP0 resulted in an increase in histone occupancy and a decrease in the ratio of acetylated histones on the viral genome during lytic infection (7). Since we have minimized ICP0 protein expression in our assays, it is unlikely that ICP0 will have bypassed the need for the VP16-dependent recruitment of coactivators in the present work.

An alternative mechanism that might lead to histone depletion is high rates of transcription by RNAP II. Several studies of yeast indicated that histones are depleted from heavily transcribed regions (19, 31, 33, 39, 61). Certain histone chaperones, such as Spt6 and FACT, are components of the RNAP II transcription machinery and facilitate elongation by RNAP II (2, 27). By extension, then, VP16, ICP0, and ICP4 might all contribute to effective transcription by RNAP II, which may in turn result in histone depletion from the viral genome. Another histone chaperone that is potentially involved in histone dynamics on the viral genome is HIRA, which is implicated in replication-independent histone deposition (18, 60) and was shown to be present in PML bodies in senescent cells (77). Future studies should test whether RNAP II or the associated histone chaperones underlie histone depletion from the HSV-1 genome.

Overall, we have shown in this report that various transcriptional coactivators are not required for IE gene expression during lytic infection or during the VP16-mediated induction of IE gene transcription from nucleosome-laden HSV-1 genomes in cultured cells. Future studies should address whether other transcriptional coactivators contribute to viral gene transcription during lytic infection. The underlying mechanism for histone deposition on or histone removal from the HSV-1 genome must still be defined. Finally, the role of chromatin dynamics during the establishment and reactivation of latent infections remains an important and incompletely answered question.


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ACKNOWLEDGMENTS
 
BRM, Brg-1, dnBRM, and dnBRG-1 expression plasmids were generously provided by Bernard Weissman and David Reisman. Plasmid pCI-p300 was obtained from Yoshihiro Nakatani. We thank Rick Hay and Wade Bresnahan for providing SW13 and telomerase-transformed human foreskin fibroblasts, respectively. We thank Carrie Graveel, Xu Lu, and Glen Alberts for comments on the manuscript.

This research was supported by the Department of Biochemistry and Molecular Biology at Michigan State University, the Van Andel Research Institute, and NIH grant AI064634. S.B.K. was supported by a special fellowship from the College of Natural Science at Michigan State University and by a predoctoral fellowship from the American Heart Association. S.L.D. was supported by a summer research internship funded by the Fred and Lena Meijer Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, MI 49503. Phone: (616) 234-5704. Fax: (616) 234-5709. E-mail: steve.triezenberg{at}vai.org Back

{triangledown} Published ahead of print on 28 January 2009. Back

{dagger} Present address: Central Michigan University, Mt. Pleasant, MI 48859. Back


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REFERENCES
 
    1
  1. Banine, F., C. Bartlett, R. Gunawardena, C. Muchardt, M. Yaniv, E. S. Knudsen, B. E. Weissman, and L. S. Sherman. 2005. SWI/SNF chromatin-remodeling factors induce changes in DNA methylation to promote transcriptional activation. Cancer Res. 65:3542-3547.[Abstract/Free Full Text]
    2. 2
  2. Belotserkovskaya, R., S. Oh, V. A. Bondarenko, G. Orphanides, V. M. Studitsky, and D. Reinberg. 2003. FACT facilitates transcription-dependent nucleosome alteration. Science 301:1090-1093.[Abstract/Free Full Text]
    3. 3
  3. Black, J. C., J. E. Choi, S. R. Lombardo, and M. Carey. 2006. A mechanism for coordinating chromatin modification and preinitiation complex assembly. Mol. Cell 23:809-818.[CrossRef][Medline]
    4. 4
  4. Boeger, H., J. Griesenbeck, J. S. Strattan, and R. D. Kornberg. 2003. Nucleosomes unfold completely at a transcriptionally active promoter. Mol. Cell 11:1587-1598.[CrossRef][Medline]
    5. 5
  5. Boyes, J., P. Byfield, Y. Nakatani, and V. Ogryzko. 1998. Regulation of activity of the transcription factor GATA-1 by acetylation. Nature 396:594-598.[CrossRef][Medline]
    6. 6
  6. Campbell, M. E., J. W. Palfreyman, and C. M. Preston. 1984. Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription. J. Mol. Biol. 180:1-19.[CrossRef][Medline]
    7. 7
  7. Cliffe, A. R., and D. M. Knipe. 2008. Herpes simplex virus ICP0 promotes both histone removal and acetylation on viral DNA during lytic infection. J. Virol. 82:12030-12038.[Abstract/Free Full Text]
    8. 8
  8. Coleman, H. M., V. Connor, Z. S. Cheng, F. Grey, C. M. Preston, and S. Efstathiou. 2008. Histone modifications associated with herpes simplex virus type 1 genomes during quiescence and following ICP0-mediated de-repression. J. Gen. Virol. 89:68-77.[Abstract/Free Full Text]
    9. 9
  9. Cress, A., and S. J. Triezenberg. 1991. Nucleotide and deduced amino acid sequences of the gene encoding virion protein 16 of herpes simplex virus type 2. Gene 103:235-238.[CrossRef][Medline]
    10. 10
  10. Deshmane, S. L., and N. W. Fraser. 1989. During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure. J. Virol. 63:943-947.[Abstract/Free Full Text]
    11. 11
  11. Flaus, A., and T. Owen-Hughes. 2004. Mechanisms for ATP-dependent chromatin remodelling: farewell to the tuna-can octamer? Curr. Opin. Genet. Dev. 14:165-173.[CrossRef][Medline]
    12. 12
  12. Goodrich, J. A., T. Hoey, C. J. Thut, A. Admon, and R. Tjian. 1993. Drosophila TAFII40 interacts with both a VP16 activation domain and the basal transcription factor TFIIB. Cell 75:519-530.[CrossRef][Medline]
    13. 13
  13. Gu, H., Y. Liang, G. Mandel, and B. Roizman. 2005. Components of the REST/CoREST/histone deacetylase repressor complex are disrupted, modified, and translocated in HSV-1-infected cells. Proc. Natl. Acad. Sci. USA 102:7571-7576.[Abstract/Free Full Text]
    14. 14
  14. Gu, H., and B. Roizman. 2007. Herpes simplex virus-infected cell protein 0 blocks the silencing of viral DNA by dissociating histone deacetylases from the CoREST-REST complex. Proc. Natl. Acad. Sci. USA 104:17134-17139.[Abstract/Free Full Text]
    15. 15
  15. Gunawardena, R. W., H. Siddiqui, D. A. Solomon, C. N. Mayhew, J. Held, S. P. Angus, and E. S. Knudsen. 2004. Hierarchical requirement of SWI/SNF in retinoblastoma tumor suppressor-mediated repression of Plk1. J. Biol. Chem. 279:29278-29285.[Abstract/Free Full Text]
    16. 16
  16. Gutierrez, J. L., M. Chandy, M. J. Carrozza, and J. L. Workman. 2007. Activation domains drive nucleosome eviction by SWI/SNF. EMBO J. 26:730-740.[CrossRef][Medline]
    17. 17
  17. Hardy, S., M. Brand, G. Mittler, J. Yanagisawa, S. Kato, M. Meisterernst, and L. Tora. 2002. TATA-binding protein-free TAF-containing complex (TFTC) and p300 are both required for efficient transcriptional activation. J. Biol. Chem. 277:32875-32882.[Abstract/Free Full Text]
    18. 18
  18. Haushalter, K. A., and J. T. Kadonaga. 2003. Chromatin assembly by DNA-translocating motors. Nat. Rev. Mol. Cell Biol. 4:613-620.[CrossRef][Medline]
    19. 19
  19. Henikoff, S. 2008. Nucleosome destabilization in the epigenetic regulation of gene expression. Nat. Rev. Genet. 9:15-26.[CrossRef][Medline]
    20. 20
  20. Herrera, F. J., and S. J. Triezenberg. 2004. VP16-dependent association of chromatin-modifying coactivators and underrepresentation of histones at immediate-early gene promoters during herpes simplex virus infection. J. Virol. 78:9689-9696.[Abstract/Free Full Text]
    21. 21
  21. Horwitz, G. A., K. Zhang, M. A. McBrian, M. Grunstein, S. K. Kurdistani, and A. J. Berk. 2008. Adenovirus small e1a alters global patterns of histone modification. Science 321:1084-1085.[Abstract/Free Full Text]
    22. 22
  22. Huang, J., J. R. Kent, B. Placek, K. A. Whelan, C. M. Hollow, P. Y. Zeng, N. W. Fraser, and S. L. Berger. 2006. Trimethylation of histone H3 lysine 4 by Set1 in the lytic infection of human herpes simplex virus 1. J. Virol. 80:5740-5746.[Abstract/Free Full Text]
    23. 23
  23. Ikeda, K., D. J. Steger, A. Eberharter, and J. L. Workman. 1999. Activation domain-specific and general transcription stimulation by native histone acetyltransferase complexes. Mol. Cell. Biol. 19:855-863.[Abstract/Free Full Text]
    24. 24
  24. Ingles, C. J., M. Shales, W. D. Cress, S. J. Triezenberg, and J. Greenblatt. 1991. Reduced binding of TFIID to transcriptionally compromised mutants of VP16. Nature 351:588-590.[CrossRef][Medline]
    25. 25
  25. Ito, T., T. Ikehara, T. Nakagawa, W. L. Kraus, and M. Muramatsu. 2000. p300-mediated acetylation facilitates the transfer of histone H2A-H2B dimers from nucleosomes to a histone chaperone. Genes Dev. 14:1899-1907.[Abstract/Free Full Text]
    26. 26
  26. Kalkhoven, E. 2004. CBP and p300: HATs for different occasions. Biochem. Pharmacol. 68:1145-1155.[CrossRef][Medline]
    27. 27
  27. Kaplan, C. D., L. Laprade, and F. Winston. 2003. Transcription elongation factors repress transcription initiation from cryptic sites. Science 301:1096-1099.[Abstract/Free Full Text]
    28. 28
  28. Kent, J. R., P. Y. Zeng, D. Atanasiu, J. Gardner, N. W. Fraser, and S. L. Berger. 2004. During lytic infection herpes simplex virus type 1 is associated with histones bearing modifications that correlate with active transcription. J. Virol. 78:10178-10186.[Abstract/Free Full Text]
    29. 29
  29. Klemm, R. D., J. A. Goodrich, S. Zhou, and R. Tjian. 1995. Molecular cloning and expression of the 32-kDa subunit of human TFIID reveals interactions with VP16 and TFIIB that mediate transcriptional activation. Proc. Natl. Acad. Sci. USA 92:5788-5792.[Abstract/Free Full Text]
    30. 30
  30. Kouzarides, T. 2007. Chromatin modifications and their function. Cell 128:693-705.[CrossRef][Medline]
    31. 31
  31. Kristjuhan, A., and J. Q. Svejstrup. 2004. Evidence for distinct mechanisms facilitating transcript elongation through chromatin in vivo. EMBO J. 23:4243-4252.[CrossRef][Medline]
    32. 32
  32. Kubat, N. J., R. K. Tran, P. McAnany, and D. C. Bloom. 2004. Specific histone tail modification and not DNA methylation is a determinant of herpes simplex virus type 1 latent gene expression. J. Virol. 78:1139-1149.[Abstract/Free Full Text]
    33. 33
  33. Kulaeva, O. I., D. A. Gaykalova, and V. M. Studitsky. 2007. Transcription through chromatin by RNA polymerase II: histone displacement and exchange. Mutat. Res. 618:116-129.[Medline]
    34. 34
  34. Kundu, T. K., V. B. Palhan, Z. Wang, W. An, P. A. Cole, and R. G. Roeder. 2000. Activator-dependent transcription from chromatin in vitro involving targeted histone acetylation by p300. Mol. Cell 6:551-561.[CrossRef][Medline]
    35. 35
  35. Kutluay, S. B., J. Doroghazi, M. E. Roemer, and S. J. Triezenberg. 2008. Curcumin inhibits herpes simplex virus immediate-early gene expression by a mechanism independent of p300/CBP histone acetyltransferase activity. Virology 373:239-247.[CrossRef][Medline]
    36. 36
  36. Kwon, Y. S., I. Garcia-Bassets, K. R. Hutt, C. S. Cheng, M. Jin, D. Liu, C. Benner, D. Wang, Z. Ye, M. Bibikova, J. B. Fan, L. Duan, C. K. Glass, M. G. Rosenfeld, and X. D. Fu. 2007. Sensitive ChIP-DSL technology reveals an extensive estrogen receptor alpha-binding program on human gene promoters. Proc. Natl. Acad. Sci. USA 104:4852-4857.[Abstract/Free Full Text]
    37. 37
  37. LaMorte, V. J., J. A. Dyck, R. L. Ochs, and R. M. Evans. 1998. Localization of nascent RNA and CREB binding protein with the PML-containing nuclear body. Proc. Natl. Acad. Sci. USA 95:4991-4996.[Abstract/Free Full Text]
    38. 38
  38. Larschan, E., and F. Winston. 2001. The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4. Genes Dev. 15:1946-1956.[Abstract/Free Full Text]
    39. 39
  39. Lee, C. K., Y. Shibata, B. Rao, B. D. Strahl, and J. D. Lieb. 2004. Evidence for nucleosome depletion at active regulatory regions genome-wide. Nat. Genet. 36:900-905.[CrossRef][Medline]
    40. 40
  40. Leinbach, S. S., and W. C. Summers. 1980. The structure of herpes simplex virus type 1 DNA as probed by micrococcal nuclease digestion. J. Gen. Virol. 51:45-59.[Abstract/Free Full Text]
    41. 41
  41. Lentine, A. F., and S. L. Bachenheimer. 1990. Intracellular organization of herpes simplex virus type 1 DNA assayed by staphylococcal nuclease sensitivity. Virus Res. 16:275-292.[CrossRef][Medline]
    42. 42
  42. Li, B., M. Carey, and J. L. Workman. 2007. The role of chromatin during transcription. Cell 128:707-719.[CrossRef][Medline]
    43. 43
  43. Lin, Y. S., I. Ha, E. Maldonado, D. Reinberg, and M. R. Green. 1991. Binding of general transcription factor TFIIB to an acidic activating region. Nature 353:569-571.[CrossRef][Medline]
    44. 44
  44. Lopez, P., R. J. Jacob, and B. Roizman. 2002. Overexpression of promyelocytic leukemia protein precludes the dispersal of ND10 structures and has no effect on accumulation of infectious herpes simplex virus 1 or its proteins. J. Virol. 76:9355-9367.[Abstract/Free Full Text]
    45. 45
  45. Melroe, G. T., L. Silva, P. A. Schaffer, and D. M. Knipe. 2007. Recruitment of activated IRF-3 and CBP/p300 to herpes simplex virus ICP0 nuclear foci: potential role in blocking IFN-beta induction. Virology 360:305-321.[CrossRef][Medline]
    46. 46
  46. Memedula, S., and A. S. Belmont. 2003. Sequential recruitment of HAT and SWI/SNF components to condensed chromatin by VP16. Curr. Biol. 13:241-246.[CrossRef][Medline]
    47. 47
  47. Muggeridge, M. I., and N. W. Fraser. 1986. Chromosomal organization of the herpes simplex virus genome during acute infection of the mouse central nervous system. J. Virol. 59:764-767.[Abstract/Free Full Text]
    48. 48
  48. Narayanan, A., W. T. Ruyechan, and T. M. Kristie. 2007. The coactivator host cell factor-1 mediates Set1 and MLL1 H3K4 trimethylation at herpesvirus immediate early promoters for initiation of infection. Proc. Natl. Acad. Sci. USA 104:10835-10840.[Abstract/Free Full Text]
    49. 49
  49. Neely, K. E., A. H. Hassan, C. E. Brown, L. Howe, and J. L. Workman. 2002. Transcription activator interactions with multiple SWI/SNF subunits. Mol. Cell. Biol. 22:1615-1625.[Abstract/Free Full Text]
    50. 50
  50. Neely, K. E., A. H. Hassan, A. E. Wallberg, D. J. Steger, B. R. Cairns, A. P. Wright, and J. L. Workman. 1999. Activation domain-mediated targeting of the SWI/SNF complex to promoters stimulates transcription from nucleosome arrays. Mol. Cell 4:649-655.[CrossRef][Medline]
    51. 51
  51. Neumann, D. M., P. S. Bhattacharjee, N. V. Giordani, D. C. Bloom, and J. M. Hill. 2007. In vivo changes in the patterns of chromatin structure associated with the latent herpes simplex virus type 1 genome in mouse trigeminal ganglia can be detected at early times after butyrate treatment. J. Virol. 81:13248-13253.[Abstract/Free Full Text]
    52. 52
  52. Oh, J., and N. W. Fraser. 2008. Temporal association of the herpes simplex virus genome with histone proteins during a lytic infection. J. Virol. 82:3530-3537.[Abstract/Free Full Text]
    53. 53
  53. Ohshima, T., T. Suganuma, and M. Ikeda. 2001. A novel mutation lacking the bromodomain of the transcriptional coactivator p300 in the SiHa cervical carcinoma cell line. Biochem. Biophys. Res. Commun. 281:569-575.[CrossRef][Medline]
    54. 54
  54. Ottosen, S., F. J. Herrera, J. R. Doroghazi, A. Hull, S. Mittal, W. S. Lane, and S. J. Triezenberg. 2006. Phosphorylation of the VP16 transcriptional activator protein during herpes simplex virus infection and mutational analysis of putative phosphorylation sites. Virology 345:468-481.[CrossRef][Medline]
    55. 55
  55. Patel, D., S. M. Huang, L. A. Baglia, and D. J. McCance. 1999. The E6 protein of human papillomavirus type 16 binds to and inhibits co-activation by CBP and p300. EMBO J. 18:5061-5072.[CrossRef][Medline]
    56. 56
  56. Reinke, H., and W. Horz. 2003. Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter. Mol. Cell 11:1599-1607.[CrossRef][Medline]
    57. 57
  57. Reisman, D. N., M. W. Strobeck, B. L. Betz, J. Sciariotta, W. Funkhouser, Jr., C. Murchardt, M. Yaniv, L. S. Sherman, E. S. Knudsen, and B. E. Weissman. 2002. Concomitant down-regulation of BRM and BRG1 in human tumor cell lines: differential effects on RB-mediated growth arrest vs CD44 expression. Oncogene 21:1196-1207.[CrossRef][Medline]
    58. 58
  58. Sadowski, I., J. Ma, S. Triezenberg, and M. Ptashne. 1988. GAL4-VP16 is an unusually potent transcriptional activator. Nature 335:563-564.[CrossRef][Medline]
    59. 59
  59. Saha, A., J. Wittmeyer, and B. R. Cairns. 2006. Chromatin remodelling: the industrial revolution of DNA around histones. Nat. Rev. Mol. Cell Biol. 7:437-447.[CrossRef][Medline]
    60. 60
  60. Sarma, K., and D. Reinberg. 2005. Histone variants meet their match. Nat. Rev. Mol. Cell Biol. 6:139-149.[CrossRef][Medline]
    61. 61
  61. Schwabish, M. A., and K. Struhl. 2004. Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 24:10111-10117.[Abstract/Free Full Text]
    62. 62
  62. Sears, A. E., V. Hukkanen, M. A. Labow, A. J. Levine, and B. Roizman. 1991. Expression of the herpes simplex virus 1 {alpha} transinducing factor (VP16) does not induce reactivation of latent virus or prevent the establishment of latency in mice. J. Virol. 65:2929-2935.[Abstract/Free Full Text]
    63. 63
  63. Stafford, G. A., and R. H. Morse. 2001. GCN5 dependence of chromatin remodeling and transcriptional activation by the GAL4 and VP16 activation domains in budding yeast. Mol. Cell. Biol. 21:4568-4578.[Abstract/Free Full Text]
    64. 64
  64. Strobeck, M. W., M. F. DeCristofaro, F. Banine, B. E. Weissman, L. S. Sherman, and E. S. Knudsen. 2001. The BRG-1 subunit of the SWI/SNF complex regulates CD44 expression. J. Biol. Chem. 276:9273-9278.[Abstract/Free Full Text]
    65. 65
  65. Strobeck, M. W., D. N. Reisman, R. W. Gunawardena, B. L. Betz, S. P. Angus, K. E. Knudsen, T. F. Kowalik, B. E. Weissman, and E. S. Knudsen. 2002. Compensation of BRG-1 function by Brm: insight into the role of the core SWI-SNF subunits in retinoblastoma tumor suppressor signaling. J. Biol. Chem. 277:4782-4789.[Abstract/Free Full Text]
    66. 66
  66. Tal-Singer, R., R. Pichyangkura, E. Chung, T. M. Lasner, B. P. Randazzo, J. Q. Trojanowski, N. W. Fraser, and S. J. Triezenberg. 1999. The transcriptional activation domain of VP16 is required for efficient infection and establishment of latency by HSV-1 in the murine peripheral and central nervous systems. Virology 259:20-33.[CrossRef][Medline]
    67. 67
  67. Uhlmann, T., S. Boeing, M. Lehmbacher, and M. Meisterernst. 2007. The VP16 activation domain establishes an active mediator lacking CDK8 in vivo. J. Biol. Chem. 282:2163-2173.[Abstract/Free Full Text]
    68. 68
  68. Utley, R. T., K. Ikeda, P. A. Grant, J. Cote, D. J. Steger, A. Eberharter, S. John, and J. L. Workman. 1998. Transcriptional activators direct histone acetyltransferase complexes to nucleosomes. Nature 394:498-502.[CrossRef][Medline]
    69. 69
  69. Vo, N., and R. H. Goodman. 2001. CREB-binding protein and p300 in transcriptional regulation. J. Biol. Chem. 276:13505-13508.[Free Full Text]
    70. 70
  70. Wang, L., S. R. Grossman, and E. Kieff. 2000. Epstein-Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter. Proc. Natl. Acad. Sci. USA 97:430-435.[Abstract/Free Full Text]
    71. 71
  71. Wang, Q. Y., C. Zhou, K. E. Johnson, R. C. Colgrove, D. M. Coen, and D. M. Knipe. 2005. Herpesviral latency-associated transcript gene promotes assembly of heterochromatin on viral lytic-gene promoters in latent infection. Proc. Natl. Acad. Sci. USA 102:16055-16059.[Abstract/Free Full Text]
    72. 72
  72. Wang, W., J. Cote, Y. Xue, S. Zhou, P. A. Khavari, S. R. Biggar, C. Muchardt, G. V. Kalpana, S. P. Goff, M. Yaniv, J. L. Workman, and G. R. Crabtree. 1996. Purification and biochemical heterogeneity of the mammalian SWI-SNF complex. EMBO J. 15:5370-5382.[Medline]
    73. 73
  73. Wong, A. K., F. Shanahan, Y. Chen, L. Lian, P. Ha, K. Hendricks, S. Ghaffari, D. Iliev, B. Penn, A. M. Woodland, R. Smith, G. Salada, A. Carillo, K. Laity, J. Gupte, B. Swedlund, S. V. Tavtigian, D. H. Teng, and E. Lees. 2000. BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines. Cancer Res. 60:6171-6177.[Abstract/Free Full Text]
    74. 74
  74. Wysocka, J., and W. Herr. 2003. The herpes simplex virus VP16-induced complex: the makings of a regulatory switch. Trends Biochem. Sci. 28:294-304.[CrossRef][Medline]
    75. 75
  75. Xiao, H., A. Pearson, B. Coulombe, R. Truant, S. Zhang, J. L. Regier, S. J. Triezenberg, D. Reinberg, O. Flores, C. J. Ingles, et al. 1994. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol. Cell. Biol. 14:7013-7024.[Abstract/Free Full Text]
    76. 76
  76. Yang, W. C., G. V. Devi-Rao, P. Ghazal, E. K. Wagner, and S. J. Triezenberg. 2002. General and specific alterations in programming of global viral gene expression during infection by VP16 activation-deficient mutants of herpes simplex virus type 1. J. Virol. 76:12758-12774.[Abstract/Free Full Text]
    77. 77
  77. Ye, X., B. Zerlanko, R. Zhang, N. Somaiah, M. Lipinski, P. Salomoni, and P. D. Adams. 2007. Definition of pRB- and p53-dependent and -independent steps in HIRA/ASF1a-mediated formation of senescence-associated heterochromatin foci. Mol. Cell. Biol. 27:2452-2465.[Abstract/Free Full Text]
    78. 78
  78. Yudkovsky, N., C. Logie, S. Hahn, and C. L. Peterson. 1999. Recruitment of the SWI/SNF chromatin remodeling complex by transcriptional activators. Genes Dev. 13:2369-2374.[Abstract/Free Full Text]
    79. 79
  79. Zimmermann, H., R. Degenkolbe, H. U. Bernard, and M. J. O'Connor. 1999. The human papillomavirus type 16 E6 oncoprotein can down-regulate p53 activity by targeting the transcriptional coactivator CBP/p300. J. Virol. 73:6209-6219.[Abstract/Free Full Text]

Journal of Virology, April 2009, p. 3436-3449, Vol. 83, No. 8
0022-538X/09/$08.00+0     doi:10.1128/JVI.02349-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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  • Kutluay, S. B., Triezenberg, S. J. (2009). Regulation of Histone Deposition on the Herpes Simplex Virus Type 1 Genome during Lytic Infection. J. Virol. 83: 5835-5845 [Abstract] [Full Text]  

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