Human Cytomegalovirus Infection Causes ... - Journal of Virology

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Apr 15, 2011 - Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School .... Phone: 82 31 299 6222. ... IE1 was deleted (15) were prepared in ihfie1.3 cells as described previously (2).
JOURNAL OF VIROLOGY, Nov. 2011, p. 11928–11937 0022-538X/11/$12.00 doi:10.1128/JVI.00758-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 85, No. 22

Human Cytomegalovirus Infection Causes Degradation of Sp100 Proteins That Suppress Viral Gene Expression䌤 Young-Eui Kim,1† Jin-Hyoung Lee,1† Eui Tae Kim,1 Hye Jin Shin,1 Su Yeon Gu,1 Hyang Sook Seol,1 Paul D. Ling,2 Chan Hee Lee,3 and Jin-Hyun Ahn1* Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon,1 and Division of Life Sciences, Chungbuk National University, Cheongju,3 Republic of Korea, and Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas2 Received 15 April 2011/Accepted 18 August 2011

The interferon-inducible Sp100 proteins are thought to play roles in the chromatin pathway and in transcriptional regulation. Sp100A, the smallest isoform, is one of the major components of PML nuclear bodies (NBs) that exhibit intrinsic antiviral activity against several viruses. Since PML NBs are disrupted by the immediate-early 1 (IE1) protein during human cytomegalovirus (HCMV) infection, the modulation of Sp100 protein expression or activity during infection has been suggested. Here, we show that Sp100 proteins are lost largely in the late stages of HCMV infection. This event required viral gene expression and involved posttranscriptional control. The mutant virus with deletion of the sequence for IE1 (CR208) did not have Sp100 loss. In CR208 infection, PML depletion by RNA interference abrogated the accumulation of SUMO-modified Sp100A and of certain high-molecular-weight Sp100 isoforms but did not significantly affect unmodified Sp100A, suggesting that the IE1-induced disruption of PML NBs is not sufficient for the complete loss of Sp100 proteins. Sp100A loss was found to require proteasome activity. Depletion of all Sp100 proteins by RNA silencing enhanced HCMV replication and major IE (MIE) gene expression. Sp100 knockdown enhanced the acetylation level of histones associated with the MIE promoter, demonstrating that the repressive effect of Sp100 proteins may involve, at least in part, the epigenetic control of the MIE promoter. Sp100A was found to interact directly with IE1 through the N-terminal dimerization domain. These findings indicate that the IE1-dependent loss of Sp100 proteins during HCMV infection may represent an important requirement for efficient viral growth. During the early stages of human cytomegalovirus (HCMV) infection, the 72-kDa immediate-early 1 (IE1 or IE72) protein targets the subnuclear structures referred to as PML nuclear bodies (NBs) (also known as nuclear domain 10 [ND10] or PML oncogenic domains [PODs]), in which input viral genomes are deposited and IE transcription occurs (22). However, the targeting of IE1 to PML NBs is transient, and subsequently, PML NBs are disrupted in an IE1-dependent manner and both IE1 and the components of PML NBs, including PML and Sp100 proteins, are relocalized into the nucleoplasm (4, 25, 27, 53). Several lines of evidence suggest that this early event promotes viral replication. The overexpression of PML conferred resistance to HCMV infection (3), and the analysis of IE1 mutants demonstrated that the ability of IE1 to disrupt PML NBs was correlated with its transactivation activity and efficient viral growth in cells transfected with HCMVbacterial artificial chromosome (BAC) DNA (30). In addition, the depletion of PML by RNA interference has been reported to promote viral replication efficiency (47). These results support the notion that PML NBs are intrinsic defense sites at which the epigenetic silencing of input viral DNA genome may

* Corresponding author. Mailing address: Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Cheoncheondong Jangangu, Suwon, Gyeonggido 440-746, Republic of Korea. Phone: 82 31 299 6222. Fax: 82 31 299 6239. E-mail: address: [email protected]. † Y.-E. Kim and J.-H. Lee equally contributed to this paper. 䌤 Published ahead of print on 31 August 2011.

take place and that the components of PML NBs perform antiviral roles against a variety of DNA and RNA viruses (for reviews, refer to references 10, 37, 48, and 49). Sp100 is a family of proteins produced by alternative splicing of a single primary transcript, which contains at least four different spliced forms: Sp100A, Sp100B, Sp100C, and Sp100HMG (8, 18, 40, 41, 45). Sp100 transcription is interferon (IFN) inducible (17), and Sp100A is predominantly localized in PML NBs, whereas only subsets of Sp100B, Sp100-HMG, and Sp100C appear to be associated with PML NBs. The direct roles of Sp100 proteins in HCMV growth have yet to be addressed, but the Sp100 proteins have been suggested to function as transcription regulators for herpesviral genes. In herpes simplex virus type 1 (HSV-1) infection, the expression of IE genes is suppressed by Sp100B, Sp100-HMG, and Sp100C but not by Sp100A (21, 34, 35, 52). Additionally, it is notable that Sp100B, Sp100-HMG, and Sp100C harbor the SAND domain (a DNA binding domain [6]) within their C-terminal regions. Sp100A has been shown to regulate the transcriptional activation function of ETS-1 in a promoter-dependent fashion (51, 55, 56) and to repress the transactivation activity of Bright, a B-cell-specific transactivator (57). Furthermore, in EpsteinBarr virus (EBV) infection, Sp100A has been determined to function as a mediator of the coactivation function of EBNA-LP in the EBNA2-mediated transactivation of viral promoters (32). Despite the potential roles played by Sp100 proteins during herpesvirus infection, the impact of HCMV infection on the

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expression of Sp100 proteins and the effects of their expression on HCMV growth have not been investigated. In this study, we assessed the expression patterns of Sp100 proteins in HCMVinfected human fibroblasts (HFs). In particular, the roles of PML NBs and their disruption by IE1 in the regulation of Sp100 expression were addressed using mutant virus from which IE1 was deleted and PML-depleted cells. Furthermore, the regulatory role of Sp100 proteins in viral gene expression and DNA replication was investigated by ablating the expression of Sp100 proteins by RNA interference. We also investigated the possible mechanisms for the Sp100-mediated suppression of viral gene expression and the direct association of IE1 with Sp100 proteins. MATERIALS AND METHODS Cells and viruses. Primary human HFs and human embryonic kidney 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a 5% CO2 humidified incubator at 37°C. The cell growth medium also contained 100 units/ml of penicillin and 100 ␮g/ml of streptomycin. Stocks for parent Towne virus and CR208 mutant virus in which IE1 was deleted (15) were prepared in ihfie1.3 cells as described previously (2). To produce UV-inactivated HCMV (UV-HCMV), the virus stock was irradiated with UV light three times at 0.72 J/cm2 using a CL-1000 cross-linker (UVP, Upland, CA). The HCMV strain AD169 was obtained from ATCC (VR-538). A clinical isolate, JHC, and the preparation of its stock were described previously (54). HCMV recombinants harboring the polymerase (Pol; UL54)-luciferase or the pp28 (UL99)-luciferase reporter construct were also previously described (3). Plasmids. Mammalian expression plasmids for wild-type or mutant Sp100A with a C-terminal HA tag were described previously (32). Plasmids for HA-IE1 (wild type and the mutant ⌬290-320) were described previously (30, 31). Plasmid for Myc-IE1 was produced by moving the cDNA on the pENTR vector (Invitrogen) into pCS3-MT (with a 6-Myc tag) plasmid (38) by using Gateway technology (Invitrogen). Saccharomyces cerevisiae yeast expression plasmids for the GAL4 DNA-binding (DB) domain/Sp100A fusion and GAL4 activation (GAL4-A) domain/Sp100A fusion proteins were generated on pAS1-CYH2 and pACTII backgrounds (5) using the Gateway technology. Plasmids for GAL4-A/ SUMO-1 and GAL4-A/IE1 were described previously (2, 5). Electroporation and DNA transfection. For electroporation of HFs, the Neon transfection system (Invitrogen) was used. For each reaction, 3 ⫻ 105 cells were suspended in 100 ␮l of resuspension (R) buffer and mixed with expression plasmids (up to 4 ␮g) in a 1.5-ml tube. After electroporation at 1,300 V and 40 ms, the cells were plated in 6-well plates or chamber slides. 293T cells were transfected via the N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid-buffered saline (BBS; Calbiochem) version of the calcium phosphate method. A mixture of plasmid and sterile H2O was mixed with CaCl2 (to a final concentration of 0.25 M) and with an equal volume of 2⫻ BBS (50 mM BBS [pH 7.0], 280 mM NaCl, 1.5 mM Na2HPO4). The mixture was kept at room temperature for 20 min and then added dropwise to cells. Production of PML- and Sp100-depleted cells using shRNA retroviral vectors. pSIREN-RetroQ (BD Biosciences) plasmids expressing short hairpin RNA (shRNA) for PML (shPML) and control shRNA (shC), which were previously used to generate siPML2 and siC cells, respectively (14, 47), were kindly provided by Thomas Stamminger (University Erlangen-Nurnberg, Erlangen, Germany). Retroviral stocks were prepared by cotransfecting 293T cells with pSIREN-RetroQ plasmid along with the packaging plasmids pHIT60 (expressing murine leukemia virus Gal-Pol) and pMD-G (expressing the envelope G protein of vesicular stomatitis virus [VSV]) (47) using Metafectene reagents (Biotex). Cell supernatants were harvested at 48 h after transfection and used to transduce HFs in the presence of Polybrene (7.5 ␮g per ml). Cells were selected with puromycin (2 ␮g per ml; Calbiochem), and the selected cells were maintained in medium containing 0.5 ␮g per ml of puromycin. Lentiviral vectors were used to generate Sp100-depleted cells. The pLKO.1TRC control expressing shC was purchased from Addgene. Lentiviral vectors (in a pLKO.1 background) expressing shRNAs for Sp100 (shSp100-A to shSp100-E, corresponding to TRCN0000019224 to TRCN0000019228, respectively) were purchased from Open BioSystems. Of these five different Sp100 shRNAs, shSp100-D was selected on the basis of the efficiency of Sp100 depletion in immunoblot assays using cotransfected cells (data not shown). The sense-strand DNA sequence of shSp100-D (hereafter shSp100) was 5⬘-CGCTAGGAAGCC

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AACAAACAA, which corresponds to a sequence common to isoforms of Sp100. Lentiviral stocks were prepared by cotransfecting 293T cells with lentiviral vectors and the packaging plasmids pCMV-⌬R8.91 (expressing the Gag-Pol, Tat, and Rev proteins of human immunodeficiency virus [HIV]) and pMD-G (expressing VSV-G), using Metafectene reagents (Biotex). Cell supernatants were harvested at 48 h after transfection. HFs were transduced by lentiviruses in the presence of Polybrene (7.5 ␮g per ml) and then selected with puromycin and maintained as described above. Antibodies. The rabbit polyclonal antibody (PAb) for Sp100 used in this study has been described previously (32). Mouse monoclonal Ab (MAb) 810R, which can detect both IE1 and IE2 proteins, was purchased from Chemicon. Mouse MAbs against IE1, p52 (encoded by UL44), and pp28 (UL99) were obtained from Virusys. Mouse MAb against ␤-actin was purchased from Sigma. Immunoblot analysis. Cells were harvested in phosphate-buffered saline (PBS). Samples were prepared by boiling cells in loading buffer, separated via SDS-PAGE, and then transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were blocked for at least 1 h in PBST (PBS plus 0.1% Tween 20 [Sigma]) containing 5% skim milk and then washed with PBST. After incubation with appropriate Abs, proteins were visualized using an enhanced chemiluminescence system (Roche) and Kodak X-ray film. Indirect immunofluorescence assay (IFA). Cells were fixed for 5 min in cold methanol and rehydrated in cold PBS. They were then incubated for 1 h with appropriate Abs in PBS at 37°C and then incubated with fluorescein isothiocyanate (FITC)-labeled or rhodamine/red X-coupled donkey immunoglobulin G (IgG; Jackson ImmunoResearch Laboratories, Inc.). FITC-labeled anti-HA IgG was used to stain HA-tagged proteins. For double labeling, Abs were incubated together. The slides were examined and photographed with a Carl Zeiss Axiophot microscope. RNA isolation and quantitative real-time reverse transcription-PCR (RTPCR). Total RNA was isolated from 2 ⫻ 105 cells using the TRIzol reagent (Invitrogen) and a MaXtract High Density tube (Qiagen). cDNAs were synthesized using the random hexamer primers in the SuperScript III system (Invitrogen). Quantitative real-time PCR was performed using SYBR green PCR core reagents (Applied Biosystems) and ABI Prism SDS software. The primers used to amplify Sp100A were 5⬘-GCTCAGGACCCCAGATTGTAC-3⬘ (forward) and 5⬘-CTAATCTTCTTTACCTGACCC-3⬘ (reverse). Proteasome activity assays. HFs were infected with virus at a multiplicity of infection (MOI) of 2 and treated for 2 h with 30 nM MG132 prior to cell harvest. Cell lysates were prepared using lysis buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40), and chymotrypsin-like proteasome activity was measured by incubating cell lysates with Suc-Leu-LeuVal-Tyr-7-amino-4-methylcoumarin (AMC) substrates (Bachem) (50 ␮M) and detecting the released AMC by fluorometry using a 380/460-nm filter set. ChIP assays. Chromatin immunoprecipitation (ChIP) assays were carried out using a kit (Upstate Biotechnology, Inc.). In brief, HCMV-infected control or Sp100-knockdown HFs (6 ⫻ 106) were fixed with 1% formaldehyde for 10 min at 24 h after infection and then lysed with a lysis buffer provided in the kit. ChIP assays were performed with 5 ␮g of anti-acetylated histone H4 (Upstate) Ab, anti-RNA polymerase II Ab, or control IgG. One-sixth of the lysates was reserved to facilitate quantitation of the DNA present in different samples prior to immunoprecipitation. Relative changes in precipitated DNA were calculated via quantitative real-time PCR with the Applied Biosystems ABI Prism SDS software. The primer sequences were 5⬘-TGGGACTTTCCTACTTGG-3⬘ (forward) and 5⬘-CCAGGCGATCTGACGGTT-3⬘ (reverse) for the major IE (MIE) promoter and 5⬘-TGTTGCAGTCACACCCGGTGC-3⬘ (forward) and 5⬘-TTAGC GTGGCCCTGAAGAGC-3⬘ (reverse) for the pp28 promoter. The PCR program used involved 40 amplification cycles (94°C for 30 s, 55°C for 30 s, and 68°C for 45 s). CoIP assays. For cotransfection assays, 293T cells (8 ⫻ 105) were harvested at 2 days after transfection and sonicated in 0.7 ml coimmunoprecipitation (CoIP) buffer (10 mM Tris-Cl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 3% glycerol, and 1% NP-40 containing protease inhibitors [Sigma]) with a microtip probe (Vibra cell; Sonics and Materials, Inc.) for 10 s (pulse on, l s; pulse off, 3 s). For infection assays, mock- or virus-infected HFs (2 ⫻ 106) were harvested at 24 h after infection and cell lysates were prepared by sonication in 1 ml of CoIP buffer (50 mM Tris-Cl [pH 7.4], 50 mM NaF, 5 mM sodium phosphate, and 0.1% Triton X-100 containing protease inhibitors [Sigma]). Cell lysates were incubated for 16 h with the appropriate Abs at 4°C. Thirty microliters of a 50% slurry of G-Sepharose (Amersham) was added and then absorbed for 1 h at 4°C. The mixture was pelleted and washed 7 times with CoIP buffer. The beads were resuspended and boiled for 5 min in loading buffer. Each sample was analyzed by SDS-PAGE and immunoblotting with appropriate Abs.

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FIG. 1. Expression patterns of Sp100 proteins in HCMV-infected fibroblasts. (A) HFs were mock infected (M) or infected with intact or UV-inactivated Towne virus at the indicated MOIs. Cell lysates were prepared at 24 h postinfection and immunoblotted with Abs for Sp100, IE1/IE2, and ␤-actin. (B) HFs were mock infected or infected with Towne virus at an MOI of 1 or 10. Total RNAs were prepared at 24 h postinfection, and the levels of Sp100A transcripts were measured by quantitative real-time RT-PCR. The results shown are the mean values and standard errors of three independent experiments. (C) HFs were mock infected or infected at an MOI of 0.2, 1, or 5. Cells were thoroughly washed after virus adsorption to remove any unbound virus. The culture supernatants were obtained at 24 h and used to administer fresh cells. Total cell lysates were prepared from initially infected cells (left 4 lanes) and from cells treated with the supernatants for 24 h (right 4 lanes) and subjected to immunoblot analysis with anti-Sp100 or anti-IE1/IE2 Abs. (D) HFs were mock infected or infected with Towne at an MOI of 10. Cell lysates were prepared at the indicated time points, and immunoblotting was performed with Abs for Sp100, IE1/IE2, p52 (UL44), pp28 (UL99), and ␤-actin. Lanes M, mock infection.

In vitro GST pulldown assays. Glutathione S-transferase (GST) and GST-IE1 were produced in Escherichia coli. Sp100A-HA was synthesized in vitro using a TNT quick-coupled transcription/translation system (Promega). E. coli extracts containing GST or GST-IE1 were incubated with GST 䡠 Bind agarose resin (Elpis Biotech) for 30 min at 4°C. After three washes with PBS, the beads were resuspended in TEN buffer (20 mM Tris [pH 7.4], 0.1 mM EDTA, 100 mM NaCl). Aliquots of in vitro-translated Sp100A-HA were mixed with GST or GST-IE1-containing beads in 500 ␮l of TEN buffer. The mixtures were then incubated for 1 h at 4°C with gentle stirring. After binding, the beads were washed five times with 1 ml NETN buffer (0.5% NP-40, 0.1 mM EDTA, 20 mM Tris [pH 7.4], 300 mM NaCl) and boiled for 5 min in SDS sample buffer. The eluted proteins were analyzed by immunoblotting. Yeast two-hybrid interaction assays. Yeast Y190 (MATa) cells were cotransformed with a plasmid expressing GAL4-DB fusion proteins (TRP1) and a plasmid expressing GAL4-A fusion proteins (LEU2). The transformants were selected on synthetic medium plates lacking both tryptophan and leucine. For rapid in-site assays for lacZ expression, a 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal) filter assay was used. For quantitative assays, ␤-galactosidase production was measured using o-nitrophenyl-␤-D-galactopyranoside (ONPG) assays. Yeast strains, media for yeast growth, methods for yeast transformation, and lacZ assays were all as described previously (2). Luciferase reporter assay. Cells were lysed using three freeze-thaw steps in 100 ␮l of 0.25 M Tris-HCl (pH 7.9) containing 1 mM dithiothreitol. Subsequent

procedures were performed as previously described (30). A TD-20/20 luminometer (Turner Designs) was used for the 10-s assay of the photons produced.

RESULTS IFN-inducible Sp100 proteins are lost in HCMV-infected cells. The expression of Sp100A, its SUMO-modified form, and other high-molecular-weight Sp100 isoforms in normal HFs has been reported (13, 14, 35). We also observed a similar pattern of Sp100 protein expression in HFs using our antiSp100 Ab (32) and using the anti-Sp100 Ab provided by Hans Will (43). To investigate whether the expression patterns of Sp100 proteins are regulated during HCMV infection, HFs were infected with wild-type or UV-inactivated Towne virus at MOIs ranging from 0.2 to 10, and immunoblot assays were performed at 24 h after infection. We found that the amount of Sp100 proteins increased at low MOIs but decreased at high MOIs in wild-type virus infection (Fig. 1A). However, unlike the inverse correlation between the amount of Sp100 proteins

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FIG. 2. Effect of CR208 virus infection on Sp100 protein levels. (A) HFs were infected with wild-type (Wt) Towne or mutant CR208 virus at an MOI of 10. Cell lysates were prepared at the indicated time points and subjected to immunoblotting using Abs for Sp100, IE1/IE2, p52, pp28, and ␤-actin. (B) Control HFs (shC) and cells expressing shRNA for PML (shPML) were mock infected or infected with CR208 at an MOI of 5. Total cell lysates were prepared at the indicated time points, and immunoblot analysis was carried out with Abs for Sp100, IE1/IE2, and ␤-actin. Lanes M, mock infection.

accumulated and the MOI used in wild-type virus infection, more Sp100 proteins accumulated at higher MOIs in UVinactivated virus infection (Fig. 1A). These results suggest that the loss of Sp100 protein at high MOIs requires viral gene expression. During wild-type virus infection, the Sp100A transcript level was markedly suppressed at an MOI of 10 compared to that at an MOI of 1 (Fig. 1B). This suggests that the regulation of Sp100 expression at high MOIs in part involves transcriptional control, probably through the efficient inhibition of type I IFN signaling by IE1 (20, 28, 29, 36). However, the results with an MOI of 10, in which the Sp100 transcript level was still comparable to that of mock infection but the protein level was significantly reduced, also suggest involvement of posttranscriptional control in the loss of Sp100 at high MOIs. In cases of a low MOI, the increase in Sp100 protein levels can be explained not only by the inefficient suppression of Sp100 transcription by IE1 in infected cells but also by the induction of Sp100 transcription by IFN signaling in uninfected cells that surround infected cells. To test it, we inoculated fresh HFs with the culture supernatants of infected cells taken at 24 h, incubated the cells for 24 h, and examined the induction of Sp100 proteins by immunoblotting. The results demonstrated that Sp100 proteins are indeed induced by the culture supernatants of infected cells (Fig. 1C), supporting the notion that the IFN-induced transcription of the Sp100 gene in uninfected cells may account for the strong induction of Sp100 protein levels at low MOIs. To evaluate more directly the accumulation of Sp100 proteins in infected cells, HFs were infected at an MOI of 10 and the amounts of Sp100 proteins in infected cells were analyzed at different time points by immunoblotting. The results showed that the amounts of SUMO-modified Sp100A and some highmolecular-weight Sp100 isoforms began to decline at as early as 6 h, the amount of unmodified Sp100A started to reduce from 12 h, and all Sp100 proteins disappeared almost entirely

at 48 and 72 h (Fig. 1D, top panel). Given the levels of IE2, p52 (an early protein encoded by UL44), and pp28 (a true late protein encoded by UL99), the transition from the early to the late phase of infection is thought to have occurred at about 24 h in this high-MOI experiment (Fig. 1D). The treatment with viral DNA polymerase inhibitors ganciclovir (GCV) and phosphonoacetic acid (PAA) partially inhibited the loss of Sp100 proteins during infection (data not shown). Collectively, our results suggest that although the levels of the SUMOmodified Sp100A and of some high-molecular-weight isoforms begin to decline from the early phase of infection, the loss of most Sp100 proteins, including unmodified Sp100A, starts from the early-phase-to-late-phase transition and occurs largely during the late phase. We observed a similar loss of Sp100 proteins during infection with Toledo, Ad169, and JHC, a clinical isolate (54), suggesting that the regulation of Sp100 expression is well conserved among different HCMV strains (data not shown). Effect of IE1 expression on Sp100 protein loss. Since the IE1-mediated disruption of PML NBs causes the displacement of Sp100 proteins into the nucleoplasm, we investigated the requirement of IE1 for the loss of Sp100 proteins using an IE1-deleted mutant virus (CR208), which has been shown to replicate in HFs at high MOIs (15). HFs were infected with Towne or CR208 virus at an MOI of 10, and the Sp100 protein levels were analyzed at different time points. Under these experimental conditions, the Sp100 proteins almost completely disappeared from 12 h in wild-type virus infection (Fig. 2A, left). However, after CR208 infection, the Sp100 protein levels markedly increased at 12 h, and this high level of Sp100 proteins was maintained throughout the late phase of infection (Fig. 2A, right). The robust increase of Sp100 protein levels observed after CR208 infection might be attributable to the absence of IE1-mediated inhibition of the type I IFN response. The expression levels of both viral early p52 protein and late pp28 protein were comparable between wild-type and CR208

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FIG. 3. Effects of proteasome inhibitors on Sp100 protein loss during HCMV infection and changes of proteasome activity in virus-infected cells. (A) HFs were mock infected or infected with Towne virus at an MOI of 2 and harvested at 24, 48, or 72 h or treated with dimethyl sulfoxide as a control, MG132 (10 ␮M), lactacystin (0.5 ␮M), or epoxomicin (0.5 ␮M) at 72 h and harvested at 96 h. Cell lysates were prepared and immunoblot assays were carried out using Abs for Sp100, IE1/IE2, p52 (UL44), pp28 (UL99), and ␤-actin. (B) Proteasome activity in cells infected with wild-type, UV-inactivated, and CR208 viruses. HFs were mock infected or infected with wild-type, UV-inactivated, or CR208 viruses at an MOI of 2 for 72 h and either left untreated (closed bars) or treated (open bars) with 30 ␮M MG132 for 2 h before measuring proteasome activities in cell lysates. (Top) Proteasome activities are indicated as mean values, and error bars represent the SDs of three independent experiments. RFU, relative fluorescence units; (bottom) cell lysates were analyzed by immunoblotting with Sp100, IE1/IE2, and ␤-actin Abs. Note that the SUMO-modified form of Sp100A was barely detectable under the cell lysis conditions employed, due to rapid deSUMOylation by SUMO proteases. Lane M, mock infection.

infections, indicating that both virus infections progressed similarly (Fig. 2A). These results indicate that the loss of Sp100 protein during HCMV infection is dependent on IE1 expression. IE1 has been shown to abrogate the covalent SUMO modification of Sp100 proteins in cotransfection assays using HeLa cells (33) and during the infection of cells stably expressing Sp100A fused with a red (mCherry) fluorescent protein (9). Consistent with these reports, we also observed that wild-type IE1 reduced the level of SUMO-modified Sp100A in transfected HFs, whereas the IE1 (⌬290-320) mutant, which is defective in PML NB disruption (30), did not do so (data not shown). Both Sp100 and PML proteins are covalently modified by SUMO and also noncovalently interact with SUMO through the so-called SUMO-interacting motif (SIM), which enables Sp100 proteins to associate with PML by using SUMO as a bridge in PML NBs (19, 26, 42). PML depletion in HFs was shown to destabilize SUMO-modified Sp100A and highmolecular-weight Sp100 isoforms (14). To determine whether the ability of IE1 to disrupt PML NBs is sufficient for the Sp100 loss, we also prepared PML-depleted HFs and examined the patterns of Sp100 expression after CR208 infection. PMLdepleted HFs and control cells were produced by transduction of cells with retroviruses expressing shRNA for PML (shPML) or control shRNA (shC) (47). The efficient reduction of PML expression in shPML cells was verified by immunoblotting using anti-PML Ab and IFA (data not shown). Consistent with the earlier reports (14, 35), we also observed that the levels of

SUMO-modified Sp100A and of high-molecular-weight Sp100 isoforms were significantly lower in shPML cells than in control cells, whereas the Sp100A level was only slightly reduced in shPML cells. This demonstrates that PML depletion alters the stability of Sp100 proteins (data not shown). When shC and shPML cells were infected with the CR208 virus at an MOI of 5, the levels of SUMO-modified Sp100A and of high-molecular-weight Sp100 isoforms were significantly lower in shPML cells than in shC cells, whereas the level of unmodified Sp100A was comparable in these cells (Fig. 2B). These results demonstrate that PML depletion resulted in the loss of SUMO-modified Sp100A and some high-molecularweight Sp100 isoforms but not unmodified Sp100A in CR208 infection. Our data suggest that although the disruption of PML NBs by IE1 affects the stability of SUMO-modified Sp100 proteins during wild-type HCMV infection, PML NB disruption is not sufficient to explain the complete loss of Sp100 (especially unmodified Sp100A) and that the other IE1-related activity probably acting directly on Sp100 may also be involved in the complete loss of Sp100. Loss of Sp100A is dependent on proteasome activity. We next investigated whether proteasome activity is involved in the loss of Sp100 proteins during HCMV infection. HCMV-infected HFs were harvested at 24, 48, 72, and 96 h postinfection or treated with proteasome inhibitors (MG132, epoxomicin, or lactacystin) at 72 h and harvested at 96 h. The results of immunoblotting demonstrated that the loss of unmodified Sp100A at 96 h was blocked effectively by the proteasome

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FIG. 4. Effect of Sp100 depletion on HCMV DNA replication and gene expression. (A) Total cell lysates were prepared from control HFs (shC) and cells expressing shRNA for Sp100 proteins (shSp100), and immunoblot analysis was performed with Abs for Sp100. The levels of ␤-actin, used as a loading control, are shown. (B) shC and shSp100 cells were mock infected or infected with Towne virus at an MOI of 1. Samples were obtained from the culture medium at 48 and 72 h postinfection, diluted, and assayed for viral titer (infectious units) using the infectious center assay (20). (C) shC and shSp100 cells were infected with Towne virus at an MOI of 3. At 24 h, cells were fixed and stained with anti-UL44 Abs. The percentages of infected cells showing only the nuclear diffuse pattern of UL44 are indicated as gray bars, whereas the percentages of cells exhibiting the nuclear focus or replication compartment patterns of UL44 on the nuclear diffuse background are indicated as black bars. (D) shC and shSp100 cells were mock infected or infected with recombinant HCMVs harboring a reporter gene (Pol-luciferase or pp28-luciferase) at an MOI of 1. Cell lysates were prepared at the indicated time points, and luciferase assays were carried out. The graphs indicate mean values, and the error bars represent the SDs of three independent experiments. Closed bars, assays in shC cells; open bars, assays in shSp100 cells. (E) Cell lysates (shC and shSp100) prepared from pp28-luciferase virus-infected cells (see panel D) were subjected to immunoblot analysis to quantify viral proteins such as IE1, IE2, p52, and pp28 with specific Abs. (F) shC and shSp100 cells were infected with CR208 virus at MOIs of 0.1 and 0.5. Samples were taken from the culture medium at 5 days postinfection, diluted, and assayed for virus titers (infectious units [IFU]).

inhibitors used, although the loss of SUMO-modified Sp100A and high-molecular-weight Sp100 isoforms was only weakly inhibited (Fig. 3A). It has previously been demonstrated that proteasome activity is required for efficient viral gene expression (24, 39, 50). However, under the experimental conditions adopted in this study, proteasome inhibitor treatment at 72 h did not affect the accumulation of the viral late protein pp28, thereby indicating that the lack of Sp100A loss is not due to the delayed progress of viral infection into the late phase (Fig. 3A). Overall, our results demonstrate that the loss of Sp100A in HCMV-infected cells requires proteasome activity, whereas the loss of other Sp100 proteins appears to be only partly dependent on proteasome activity. Since the degradation of unmodified Sp100A required proteasome activity, we also examined whether the absence of Sp100 loss during CR208 infection is associated with reduced proteasome activity. HFs were infected with wild-type, UVinactivated, or CR208 virus at an MOI of 2 for 72 h, and the proteasome activities in infected cell lysates were then measured. It was found that proteasome activity was increased by wild-type virus but not by UV-inactivated virus and that proteasome activity was increased in CR208-infected cells as well as in wild-type virus-infected cells (Fig. 3B, graph). Immuno-

blot assay results showed that the Sp100 proteins were already lost at 72 h in wild-type virus-infected cells, whereas unmodified Sp100A was accumulated in UV-inactivated or CR208infected cells (Fig. 3B, bottom). Note that to measure proteasome activity, the cell lysates were prepared without boiling them in protein sample buffer, and therefore, SUMO-modified Sp100A was not well preserved in cell lysates (Fig. 3B, bottom). Collectively, these observations suggest that the failure of Sp100A degradation in CR208 infection is not attributable to reduced proteasome activity. Sp100 protein depletion facilitates HCMV DNA replication and viral gene expression. To study the possible role of Sp100 proteins in the regulation of HCMV growth, we prepared Sp100-depleted HFs and control cells using lentiviruses expressing shRNA for Sp100 (shSp100) or control shRNA (shC). The expression of Sp100 proteins was effectively repressed in shSp100 cells but not in control (shC) cells in immunoblot analysis using anti-Sp100 Ab (Fig. 4A) and IFA (data not shown). Consistent with a previous report (12), Sp100 depletion did not affect the expression of PML proteins (data not shown). To measure the effect of Sp100 depletion on HCMV replication efficiency, shSp100 cells and control shC cells were in-

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fected with Towne virus at an MOI of 1, and the virus titers in culture media were determined at 48 and 72 h postinfection. We found that the virus titers were higher in shSp100 cells (by 12-fold at 48 h and by 5-fold at 72 h) than in control cells (Fig. 4B). We also compared the formation of viral replication foci or replication compartments between shC and shSp100 cells. When cells were infected with Towne virus at an MOI of 3 and stained with anti-UL44 Ab at 24 h after infection, we found that the population of cells exhibiting the UL44-positive nuclear foci or replication compartments among total UL44positive cells (including cells showing only the nuclear diffuse pattern of UL44) was higher by 2.4-fold in shSp100 cells than in shC cells (Fig. 4C). We next infected these HFs with recombinant HCMV harboring the polymerase (UL54)-luciferase or the pp28 (UL99)luciferase reporter gene (3) at an MOI of 1 and determined the UL54 and pp28 promoter activities by measuring luciferase activities. The results showed that the activation levels of these viral early and late promoters were higher in shSp100 cells than in shC cells, with a maximum 8-fold higher level observed at 36 h for the polymerase promoter and a maximum 6-fold higher level observed at 48 h for the pp28 promoter (Fig. 4D). In addition, the levels of accumulation of viral IE (IE1 and IE2), early (p52, encoded by UL44), and late (pp28, encoded by UL99) proteins in HCMV(pp28-Luc) virus-infected cells were analyzed by immunoblotting. It was determined that the accumulation of all of viral IE, early, and late proteins was higher in shSp100 cells than in control cells (Fig. 4E). We also determined that Sp100 knockdown still enhances the replication efficiency of CR208 virus (Fig. 4F). All together, these findings support the idea that, like PML, Sp100 proteins may also perform a role in intrinsic defense against HCMV by regulating viral gene expression. We further investigated the possible mechanism by which Sp100 proteins repress viral gene expression. As Sp100 proteins are thought to regulate the chromatin pathway and transcription, we measured changes in the histone acetylation levels on the viral MIE and pp28 promoters in normal and Sp100-knockdown cells. shC and shSp100 cells were infected with HCMV at an MOI of 2, and ChIP assays were performed at 24 h after infection. The results demonstrated that the levels of acetylated histone H4 on the MIE promoter were higher in Sp100-knockdown cells than in control cells, whereas the levels of acetylated histone H4 on the pp28 promoter were comparable (Fig. 5). As controls, the levels of RNA polymerase II associated with the MIE and pp28 promoters were higher in shSp100 cells than in shC cells (Fig. 5). These results demonstrate that the Sp100 depletion specifically enhances the acetylation level of histones associated with the MIE promoter. Sp100A interacts directly with IE1 through the N-terminal dimerization domain. In an attempt to gain insight into the role of IE1 in Sp100 degradation, we assessed the possible interaction between IE1 and Sp100A. First, we observed that IE1 interacts with Sp100A in yeast two-hybrid interaction assays more weakly than the Sp100–SUMO-1 interaction, but with a strength similar to that of Sp100 self-interaction (Fig. 6A). The association of IE1 with Sp100A and some highmolecular-weight Sp100 isoforms was also observed by Co-IP assays with cell extracts prepared 24 h after infection (Fig. 6B). We also mapped the region of Sp100A responsible for IE1

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FIG. 5. Effects of Sp100 depletion on the levels of acetylated histones associated with the MIE and pp28 promoters. shC and ShSp100 cells were infected with Towne virus at an MOI of 2, and ChIP assays were carried out at 24 h using acetylated histone H4-specific Ab (␣AcH4) and RNA polymerase II Ab (␣Pol II). The amounts of coprecipitated DNA were quantified by real-time PCR and normalized to the input amount, and their changes are shown as graphs.

binding by carrying out Co-IP assays in cells cotransfected with IE1 and a series of deletion constructs of Sp100A. IE1 was shown to interact with ⌬153-286, ⌬287-333, ⌬334-407, and ⌬408-480 Sp100A mutants as well as wild-type protein but did not bind to the ⌬3-152 mutant (Fig. 6C and D). The ⌬3-152 mutant contains a deletion of the N-terminal region that is required for dimerization and PML NB targeting (44). To test whether or not IE1 interacts directly with Sp100A, in vitro GST pulldown assays were carried out. The results demonstrated that bacterially expressed GST-IE1 interacts specifically with wild-type Sp100A but not with the ⌬3-152 mutant, both of which were synthesized by in vitro transcription/translation reactions. These results demonstrate that IE1 interacts directly with Sp100A through the N-terminal dimerization domain (Fig. 6E). DISCUSSION PML NBs and their components are believed to perform important roles in intrinsic host defense against infection with many DNA and RNA viruses (49). Therefore, viruses are expected to exploit and carry out countermeasures for this intrinsic host defense mechanism. Indeed, many herpesviruses are capable of disrupting PML NBs using different mechanisms. In HSV-1 infection, ICP0 targets PML for ubiquitindependent proteasomal degradation (7, 11, 16). BZLF-1 of EBV disrupts PML NBs by reducing the level of SUMOmodified PML proteins (1). Similarly, HCMV IE1 disrupts

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FIG. 6. Interaction of IE1 with Sp100A. (A) Interaction of IE1 with Sp100 in yeast cells. The X-Gal filter assays were performed using yeast cells expressing both GAL4-DB/Sp100A fusion protein and GAL4-A/SUMO-1, GAL4-A/IE1, or GAL4/Sp100A fusion protein. The cells expressing GAL4-DB/Sp100A and GAL4-A were used as a negative control. The top three panels show positive interactions. The relative strength of each interaction was also shown by directly measuring ␤-galactosidase activity in yeast cell lysates. (B) HFs were mock infected or infected with Towne virus at an MOI of 2. Total cell lysates were prepared at 24 h postinfection and immunoprecipitated with anti-IE1 Ab (ch443) or control IgG as indicated, followed by immunoblotting (IB) with anti-Sp100 and anti-IE1 Abs. Total cell lysates were also immunoblotted with anti-IE1 and anti-Sp100 Abs to show the protein expression levels. (C) 293T cells were cotransfected with plasmids encoding Sp100A-HA (wild type or mutants) and Myc-IE1, as indicated. At 48 h, total cell lysates were prepared and immunoprecipitated with anti-Myc Ab, followed by immunoblotting with anti-HA Ab. Total cell lysates were also immunoblotted with anti-Myc or anti-HA Abs. (D) Structures of wild-type and mutant Sp100A proteins used. The positions for the regions responsible for dimerization, PML NB binding, HP1␣ binding, and the nuclear localization signal (NLS) are designated. All Sp100A constructs except for ⌬408-480 harbor an HA tag at the C terminus, and ⌬408-480 harbors both an NLS tag (open circle) and an HA tag at its C terminus. (E) In vitro GST pulldown assays. Bacterially purified GST or GST-IE1 immobilized to glutathione-Sepharose beads was incubated with in vitro-translated wide-type or ⌬3-152 mutant Sp100A proteins (with the HA tag), and the bound proteins were fractionated on SDS–8% polyacrylamide gels and visualized by immunoblotting with anti-HA Ab. One-tenth of the Sp100A proteins used in each binding reaction were loaded as input controls. One-tenth of the GST or GST-IE1 proteins used in each reaction were visualized with Coomassie blue staining.

PML NBs by inducing the loss of PML SUMOylation (30, 33), which is a critical PML modification for the formation of PML NBs. The underlying mechanism appears to involve the inhibition of PML oligomerization by IE1 (23). In the present study, we demonstrate that IFN-inducible Sp100 proteins are efficiently lost in HCMV-infected HFs, largely during the late stage of infection. Furthermore, our analysis using UV-inactivated virus and mutant virus with IE1 deletion (CR208) demonstrates that the Sp100 loss occurs in an IE1-dependent manner and involves a posttranscriptional process, as well as

transcriptional regulation. Our data also show that proteasome activity is required for the degradation of Sp100A. A similar loss of SUMO-modified Sp100A and high-molecular-weight Sp100 isoforms has been reported during the early phase of HSV-1 infection (14) and in ICP0-expressing cells (13). We also noted that all Sp100 proteins are lost at the late phase of HSV-1 infection (data not shown). Therefore, the regulation of Sp100 protein expression may be a process shared by both HSV-1 and HCMV. Previous studies using PML NB-depleted cells have demon-

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strated that PML proteins are required for the stable expression of SUMO-modified Sp100A and certain high-molecularweight Sp100 isoforms (14) or all of the Sp100 proteins, including unmodified Sp100A (35). Besides being covalently modified by SUMO, both PML and Sp100 proteins also contain a SUMO-interacting motif (SIM). Therefore, the reduction of Sp100 stability in PML-depleted cells may be attributed to the lack of a stable association between Sp100 proteins and PML proteins via a SUMO bridge. We also observed that PML depletion causes an apparent loss of SUMO-modified Sp100A and some high-molecular-weight Sp100 isoforms and a slight reduction in unmodified Sp100A in HFs and that IE1 transfection results in a complete loss of Sp100A SUMOylation in cotransfection assays (data not shown). Therefore, PML NB disruption by IE1 during infection appears to contribute to the reduction of SUMO-modified Sp100. This scenario is also supported by our finding that, even in CR208 infection, PML depletion resulted in a loss of the SUMO-modified forms of Sp100A and probably of other high-molecular-weight Sp100 isoforms. Notably, after CR208 infection, a high-level accumulation of unmodified Sp100A was still observed in PML-depleted cells. This suggests that in addition to PML NB disruption, another IE1-dependent activity is required for the complete loss of all Sp100 proteins observed in wild-type virus infection. Interestingly, we found that Sp100 interacts directly with IE1 through the N-terminal dimerization domain. This interaction may inhibit Sp100 self-interaction, rendering Sp100 proteins more susceptible to proteases. Since treatment with proteasome inhibitors efficiently blocks the loss of Sp100A, this activity may be related to the targeting of Sp100A to the proteasome. Considering the case of PML degradation by HSV-1 ICP0, whether IE1 recruits an ubiquitin E3 ligase to Sp100A for proteasomal degradation would be an intriguing question. Less clear, however, is the issue of whether or not SUMO-modified Sp100 proteins are also degraded by the proteasome. Although an ubiquitin E3 ligase that specifically recognizes SUMO-modified PML substrates has recently been identified in animal cells (46), the effects of proteasome inhibitors on the loss of these SUMO-modified Sp100 proteins were found to be relatively weak. The results of this study demonstrate that the depletion of all Sp100 proteins in HFs enhances HCMV replication and gene expression, including MIE gene expression. As PML has been shown to suppress HCMV gene expression (3, 47), the effect of Sp100 depletion on HCMV gene expression may be caused indirectly via an alteration in PML function. However, consistent with the results of a previous study (12), we observed that Sp100 depletion did not influence the expression patterns of PML isoforms (data not shown). In the data from our ChIP assays, the depletion of all Sp100 proteins enhanced the acetylation level of histone H4 associated with the MIE promoter, suggesting that the repressive effect of Sp100 proteins may involve, at least in part, the epigenetic regulation of IE gene expression. In a similar experiment with HSV-1 infection, Sp100 depletion was found to increase the replication efficiency of an ICP0-null mutant virus, but not the replication efficiency of the wild-type virus (12). The reduction of the levels of SUMO-modified forms of Sp100 proteins, which results from the IE1-induced PML NB disruption, would cer-

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tainly benefit MIE gene expression. However, the physiological role of the complete loss of Sp100 proteins shown at late times is not clear. Given that the functions of Sp100 proteins are associated with the chromatin pathway and transcription, the complete loss of Sp100 proteins may regulate viral gene expression at late times or affect the chromatinization status of viral DNA. It has been reported that Sp100B, Sp100C, and Sp100-HMG repress the HSV-1 ICP0 and ICP4 promoters in reporter assays (35). In that study, Sp100 proteins were not shown to exert any effects on the activation of the HCMV MIE promoters. Understanding the specific roles of Sp100 isoforms in HCMV infection should be a specific focus of future studies. ACKNOWLEDGMENTS We express our gratitude to Thomas Stamminger and Hans Will for supplying the retroviral vector for PML shRNA and Sp100 Ab, respectively. We also thank Kyeong Ho Lee for providing us with the packaging plasmids for lentivirus production. This work was supported by the Basic Science Research Program (KRF-2007-313-C00544), the Mid-Career Research Program (20090078805), and the Ubiquitome Research Program (2011-0002136) through the National Research Foundation of South Korea (NRF) funded by the Ministry of Education, Science, and Technology. REFERENCES 1. Adamson, A. L., and S. Kenney. 2001. Epstein-Barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies. J. Virol. 75:2388–2399. 2. Ahn, J. H., E. J. Brignole III, and G. S. Hayward. 1998. Disruption of PML subnuclear domains by the acidic IE1 protein of human cytomegalovirus is mediated through interaction with PML and may modulate a RING fingerdependent cryptic transactivator function of PML. Mol. Cell. Biol. 18:4899– 4913. 3. Ahn, J. H., and G. S. Hayward. 2000. Disruption of PML-associated nuclear bodies by IE1 correlates with efficient early stages of viral gene expression and DNA replication in human cytomegalovirus infection. Virology 274:39–55. 4. Ahn, J. H., and G. S. Hayward. 1997. The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PMLassociated nuclear bodies at very early times in infected permissive cells. J. Virol. 71:4599–4613. 5. Ahn, J. H., Y. Xu, W. J. Jang, M. J. Matunis, and G. S. Hayward. 2001. Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9. J. Virol. 75:3859–3872. 6. Bottomley, M. J., et al. 2001. The SAND domain structure defines a novel DNA-binding fold in transcriptional regulation. Nat. Struct. Biol. 8:626–633. 7. Boutell, C., S. Sadis, and R. D. Everett. 2002. Herpes simplex virus type 1 immediate-early protein ICP0 and is isolated RING finger domain act as ubiquitin E3 ligases in vitro. J. Virol. 76:841–850. 8. Dent, A. L., et al. 1996. LYSP100-associated nuclear domains (LANDs): description of a new class of subnuclear structures and their relationship to PML nuclear bodies. Blood 88:1423–1426. 9. Dimitropoulou, P., et al. 2010. Differential relocation and stability of PMLbody components during productive human cytomegalovirus infection: detailed characterization by live-cell imaging. Eur. J. Cell Biol. 89:757–768. 10. Everett, R. D., and M. K. Chelbi-Alix. 2007. PML and PML nuclear bodies: implications in antiviral defence. Biochimie 89:819–830. 11. Everett, R. D., et al. 1998. The disruption of ND10 during herpes simplex virus infection correlates with the Vmw110- and proteasome-dependent loss of several PML isoforms. J. Virol. 72:6581–6591. 12. Everett, R. D., C. Parada, P. Gripon, H. Sirma, and A. Orr. 2008. Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100. J. Virol. 82:2661–2672. 13. Everett, R. D., M. L. Parsy, and A. Orr. 2009. Analysis of the functions of herpes simplex virus type 1 regulatory protein ICP0 that are critical for lytic infection and derepression of quiescent viral genomes. J. Virol. 83:4963– 4977. 14. Everett, R. D., et al. 2006. PML contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by ICP0. J. Virol. 80:7995–8005. 15. Greaves, R. F., and E. S. Mocarski. 1998. Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant. J. Virol. 72:366– 379.

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