Functional Interaction between Class II Histone Deacetylases and ICP0 of Herpes Simplex Virus Type 1

This study describes the physical and functional interactionsbetween ICP0 of herpes simplex virus type 1 and class II histonedeacetylases (HDACs) 4, 5, and 7. Class II HDACs are mainlyknown for their participation in the control of cell differentiationthrough the regulation of the activity of the transcriptionfactor MEF2 (myocyte enhancer factor 2), implicated in muscledevelopment and neuronal survival. Immunofluorescence experimentsperformed on transfected cells showed that ICP0 colocalizeswith and reorganizes the nuclear distribution of ectopicallyexpressed class I and II HDACs. In addition, endogenous HDAC4and at least one of its binding partners, the corepressor proteinSMRT (for silencing mediator of retinoid and thyroid receptor),undergo changes in their nuclear distribution in ICP0-transfectedcells. As a result, during infection endogenous HDAC4 colocalizeswith ICP0. Coimmunoprecipitation and glutathione S-transferasepull-down assays confirmed that class II but not class I HDACsspecifically interacted with ICP0 through their amino-terminalregions. This region, which is not conserved in class I HDACsbut homologous to the MITR (MEF2-interacting transcription repressor)protein, is responsible for the repression, in a deacetylase-independentmanner, of MEF2 by sequestering it under an inactive form inthe nucleus. Consequently, we show that ICP0 is able to overcomethe HDAC5 amino-terminal- and MITR-induced MEF2A repressionin gene reporter assays. This is the first report of a viralprotein interacting with and controlling the repressor activityof class II HDACs. We discuss the putative consequences of suchan interaction for the biology of the virus both during lyticinfection and reactivation from latency.

INTRODUCTION

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Introduction

The role of acetylation in the transcription pathway has beenwidely studied and is generally considered a major step towardgene expression (1). Two classes of enzymes with antagonistactivities, called histone acetyltransferases (HATs) and deacetylases(HDACs), significantly contribute to the control of expressionby the modification of the acetylation state of predominantly,but not exclusively, histone proteins (5, 70). Whereas HAT activity,and thus acetylation of histones, is generally associated withtranscription, HDAC activity, and thus deacetylation, is responsiblefor repression (69). Three groups of HDACs, defined as classesI, II, and III, have been described in higher eukaryotes, accordingto sequence similarities with yeast RPD3, HDA1 and SIR2 proteins,respectively (reviewed in references 43 and 73). Class II HDACshave been classified as such mainly on the basis of amino acidsequence homologies with the deacetylation domain of membersof the class I family. However, for most of them, and unlikeclass I HDACs, clear evidence concerning their direct implicationin deacetylation of substrates is lacking. It is now thoughtthat, with the exception of HDAC6, class II HDACs acquire theirdeacetylation activity only through the interaction of theircarboxy-terminal end with at least the class I HDAC3 and thenuclear receptor corepressor SMRT (for silencing mediator ofretinoid and thyroid receptor) (25, 26, 39, 80). Therefore,the contribution of class II HDACs to the regulation of theactivity of gene promoters is suggested by their identificationas components of multiprotein nuclear complexes known as Sin3/HDACand NuRD/Mi2/NRD (nucleosome remodeling histone deacetylasecomplex), which are implicated in repression of gene expression(reviewed in reference 44). However, class II HDAC4, -5, and-7 possess an additional repressor domain in the amino-terminalextension of the sequence that is independent of the HDAC3/SMRT-associatedcatalytic deacetylase activity (42, 46, 56, 75, 82). This domaininteracts with and represses the activity of the transcriptionfactor myocyte enhancer factor 2 (MEF2), which is involved inthe differentiation and/or survival of muscles and neurons (27,54, 59, 77). It is now clear that class II HDACs play a significantrole in the control of these processes by maintaining MEF-2under an inactive form in the nucleus (reviewed in reference73).

Herpes simplex virus type 1 (HSV-1) is a major human pathogenthat is characterized by an outstanding mode of infection madeof permanent switches between lytic cycles and latency. Aftermultiplication of the virus in epithelial cells, HSV-1 infectsneurons and delivers its 152-kb double-stranded linear DNA tothe nucleus. Under certain conditions it starts an abortiveacute infection involving limited replication of the viral genomeand then enters latency until reactivation after appropriatestimuli. One of the major differences between lytic cycle andlatency is encountered at the level of gene expression. Indeed,whereas during acute lytic infection the entire viral genomeis expressed, during latency the genome undergoes an almost-completegene expression shutoff (reviewed in references 61 and 74).

HSV-1 virion is composed of a capsid containing the genome surroundedby an envelope, both nucleocapsid and envelope being separatedby the tegument. About 80 temporarily regulated genes are presentin the viral genome. Genes and gene products are subdividedinto three major families—immediate-early (IE), early,and late—depending on the time course of synthesis andrequirement for prior viral gene expression and DNA replication(24). ICP0 is one of the five IE proteins encoded by the virus.It is a RING finger zinc-binding protein capable of activatinggene expression, from viral and cellular promoters, withoutbinding DNA (7, 12). Viruses unable to synthesize functionalICP0 are impaired in their ability to initiate a productivelytic infection, to reactivate from latently infected neurons,and to have a higher propensity to enter a nonreplicating quiescentstate after infection of cultured cells at a low multiplicity(6, 8, 14, 17, 29, 33, 34, 45, 63, 67, 68). The tendency ofICP0-deficient viruses to be silenced suggests that a cellularrepressor mechanism could control HSV-1 genome transcriptionand/or replication, and ICP0 could interfere with this mechanism.The control of the stability of a putative repressor proteinby ICP0 became a growing postulate after the discovery thatICP0 was inducing the degradation of several cellular proteinsthrough active ubiquitin-proteasome pathway (reviewed in reference18). Several recent studies described an E3 ubiquitin ligaseactivity associated with ICP0, thus confirming the direct involvementof ICP0 in this degradation pathway (3, 4, 32, 71). However,ICP0 also interacts with several cellular components withoutinducing their degradation (reviewed in reference 18), and itis likely that these interactions will account for its contributionto lytic infection and reactivation from latency. Indeed, quiescentviruses can be reactivated by later expression of ICP0 fromexternal sources or, but with less efficiency, by the additionof the inhibitor of HDACs, trichostatin A (TSA) (23, 35, 36,61, 64, 67). Based on these data, we investigated the putativephysical and functional interactions between ICP0 and classI or class II HDACs. We demonstrate that ICP0 specifically interactswith the amino-terminal region of class II HDAC4, -5, and -7,resulting in the offset of the repression activity associatedwith this domain. Consequently, the expression of ICP0 can counteractthe repressor activity of HDAC5 and the class II HDACs-relatedMITR (for MEF2-interacting transcription repressor) proteinon MEF2A, therefore enhancing significantly its transcriptionalactivity. However, unlike TSA, ICP0 does not inhibit the globaldeacetylation activity in the cell, which highlights a majordifference between ICP0 and TSA concerning mechanisms implicatedin the reactivation of quiescent viruses.

MATERIALS AND METHODS

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Materials and Methods

Viruses, cells, and preparation of cell extracts. The HSV-1 strain 17 syn+ (17+) is the parental viral strain.ICP0 mutant virus vFXE and the D30EBA virus were described previously(17, 60). HeLa cells were grown at 37°C in Glasgow modifiedEagle medium containing 10 U of penicillin/ml and 100 µgof streptomycin/ml and supplemented with 10% fetal bovine serum.For cell extracts, cells were washed once with phosphate-bufferedsaline (PBS), scraped off the flask, and then centrifuged at1,000 x g for 5 min. The cell pellet was resuspended in 200µl of a lysis buffer containing 15 mM Tris-HCl (pH 7.5),2 mM EDTA, 0.25 mM EGTA, 15 mM NaCl, 0.3 mM sucrose, 0.5% TritonX-100, and 300 mM KCl. Samples were incubated on ice for 30min and centrifuged at 6,000 x g for 5 min at 4°C to removeall debris. For glutathione S-transferase (GST) pull-down assays,400 µl of lysis buffer without KCl, to obtain a 100 mMKCl final concentration, was added to the supernatant. For immunoprecipitation,supernatants were flowed through Micro Bio-Spin 6 chromatographycolumns (Bio-Rad) preequilibrated with a buffer containing 20mM HEPES (pH 7.9), 0.2 mM EDTA, 100 mM KCl, and 25% glycerol.The protease inhibitors phenylmethylsulfonyl fluoride and proteaseinhibitor cocktail tablets (Roche) were added to the lysis buffers.Protein concentrations were determined by the Bradford method.

Chromatin-associated protein extraction was done by scrapingcells off the plates, resuspending them in PBS-phenylmethylsulfonylfluoride, and sonicating them in a ultrasonic water bath fortwice for 15 s each time. Nuclei and debris were centrifugedat 12,000 x g for 10 min at 4°C. Pellets were resuspendedin HCl (250 mM) and incubated on ice for 30 min before centrifugationat 12,000 x g for 20 min at 4°C. Trichloroacetate was addedto the supernatant to a final volume of 20%, and incubationwas carried out for another 30 min on ice before centrifugationat 12,000 x g for 20 min at 4°C. Pellets were washed oncewith 500 µl of a mixed acetone-HCl (10 mM) solution andonce with acetone alone and then dried in a SpeedVac (Savant)for 5 min before resuspension in water and dosage.

DNA constructs. Plasmids pCi110 and pCiFXE express ICP0 and its RING fingermutant FXE, respectively, from a pCineo-based plasmid (21).Hemagglutinin (HA)- and FLAG-tagged HDACs, GAL4-HDAC5 fusionprotein, pCMVßgal, pcDNAGAL4-DB, L8G5-Luc, and LEXA-VP16expressing plasmids have already been described (46, 76). GAL4-and GST-HDAC3 fusion protein-expressing plasmids were constructedby inserting the EcoRI and BamHI fragment from the pCEP4F-HD3plasmid (76) containing the entire HDAC3 gene in frame withthe tag in the pcDNAGAL4 and pGEX4T1 (Pharmacia) plasmids, respectively.

Fusion protein GST-ICP0(1-241) expressing plasmid has been describedearlier (55). GST-ICP0(1-109)- and GST-ICP0(106-241)-expressingplasmids were constructed by cloning PCR fragments obtainedwith the primers 5'-CGCGGATCCCCATGGAGCCCCGCCCCGGA-3' and 5'-CGGAATTCTACCCCCCGTCCTCTCGAG-3'for the former and 5'-CGCGGATCCCCGAGGACGGGGGGAGCGACGA-3' and5'-CGGAATTCTAGTCGTCCAGGTCGTCGTCAT-3' for the latter. After digestionby BamHI and EcoRI, fragments were cloned into the BamHI andEcoRI site of the vector pGEX-3X (Pharmacia). Both constructswere verified by sequencing. GST-ICP0(245-518) and GST-ICP0(553-775)were constructed by cloning in pGEX-3X the fragment SnaBI-MluIfor the former and AatII-HpaI for the latter, retrieved fromthe pJR3-ICP0 gene-containing plasmid (19). Constructs expressingGST-HDAC4(1-650), -HDAC5, and -HDAC7 have already been described(42, 46, 56). GST-HDAC4(569-1081) was constructed by cloningin pGEX-5X-2, a XhoI fragment obtained from the HA-tagged HDAC4plasmid. GST-HDAC3(1-429) has been constructed by insertinga XhoI fragment from GAL4-HDAC3 in a pGEX-4T1 plasmid. Expressionand purification of GST fusion proteins was done as describedpreviously (65). All of the constructs were analyzed for proteinexpression by Coomassie staining and Western blotting with anti-GST(Sigma) or anti-GAL4 (Clontech) monoclonal antibodies (MAbs).HDAC4, -5, and -7 are of mouse origin, and HDAC3 is of humanorigin.

GST pull-down assays. A total of 50 µg of GST fusion protein preparation wasmixed with lysis buffer (100 mM KCl) containing 1 mg of proteinfrom cellular extracts or 5 µl of the sample from in vitro-synthesizedproteins. All extracts were first incubated for 1 h at 4°Cwith continuous mixing with beads linked to the GST proteinexpressed from vector plasmid pGEX-3X in order to reduce thebackground signal. The precleared extracts were then incubatedwith the appropriate GST fusion protein beads and a negativeGST bead control for 2 h at room temperature with continuousmixing. The beads were harvested by brief centrifugation andthen washed five times with 500 µl of the lysis buffer(100 mM KCl). Protein complexes were directly eluted from thebeads by adding Laemmli buffer and then boiled.

Immunoprecipitations. HeLa cells were seeded at 1.2 x 10⁶ cells per 60-mm petri dish.The following day, cells were transfected with the adequateplasmids. Extracts from transfected HeLa cells were then preparedas described above and preincubated 1 h at 4°C with proteinA-Sepharose beads (Amersham) to decrease nonspecific binding.Samples were then centrifuged, and the supernatants were incubatedwith the appropriate antibodies for 1 h at 4°C. Then, 50µl of a 50% slurry protein A-Sepharose preequilibratedwith the same extract-containing buffer was added to the samples,followed by incubation for another hour. The samples were thencentrifuged briefly and washed five times with buffer beforethe beads were suspended in Laemmli buffer and then boiled.

siRNA experiments. HeLa cells were seeded at 2 x 10⁵ cells per well in six-wellLinbro multiwell plates and transfected (Effectene transfectionkit; Qiagen) the following day with 840 ng of small interferingRNA (siRNA). After 48 h, the cells were scraped off the platesin PBS buffer before protein dosage by the Bradford method,the addition of Laemmli buffer, and Western blotting. The characteristicsof the siRNAs used in the present study are as follows: siRNAac4_337,5'-GCAACAACAGGAGAUGCUGtt-3' (sense) and 5'-CAGCAUCUCCUGUUGUUGCtt-3'(antisense) (target sequence in human HDAC4 gene, nucleotides337 to 357); siRNAac4_1513, 5'-CAAGAUCAUCCCCAAGCCAtt-3' (sense)and 5'-UGGCUUGGGGAUGAUCUUGtt-3' (antisense) (target sequencein human HDAC4 gene, nucleotides 1513 to 1533); and siRNAac4_2665,5'-CGUCAACAUGGCUUUCACCtt-3' (sense) and 5'-GGUGAAAGCCAUGUUGACGtt-3'(antisense) (target sequence in human HDAC4 gene, nucleotides2665 to 2685). The siRNA for human lamin A/C has been previouslydescribed (16).

Western blotting. Samples from GST pull-down, immunoprecipitation, and siRNA assayswere treated for Western blotting according to a protocol describedearlier (48). The primary antibodies were MAbs 11060 anti-ICP0(1/10,000), 12CA5 anti-HA-1 (Roche), or M5 anti-FLAG (Sigma)epitopes (1 µg/ml), Do-7 anti-p53 (1/100; Dako), and rabbitpolyclonal sera, ML-19 anti-human HDAC4 (a generous gift fromSigma), anti-lamin A/C (Santa Cruz), anti-actin (Sigma), anti-acetylatedhistone H4 (AcH4; Santa Cruz), and anti-acetylated lysine (1µg/ml; AcLys; Cell Signaling).

In vitro protein synthesis. Plasmid pT7110 containing ICP0 encoding cDNA (22) was used forin vitro synthesis of [³⁵S]methionine-labeled ICP0 by usinga rabbit reticulocyte lysate system (TNT Quick-Coupled Transcription/TranslationSystem; Promega) according to the manufacturer's recommendations.Of the 50-µl final volume, 5 µl was used per GSTpull-down assay. Pull-down assays were performed as describedabove by adjusting sample volumes to 500 µl with the lysisbuffer (100 mM KCl). Samples were then loaded onto a sodiumdodecyl sulfate-polyacrylamide gel and electrophoresed. Gelswere then incubated for 10 min in fixing solution (50% methanol,10% acetic acid) and for 10 min in soaking solution (7% methanol,7% acetic acid, 11% glycerol). Gels were dried at 80°C undervacuum for 30 min and then exposed to film.

GAL4 binding assay. HeLa cells were seeded at 1.2 x 10⁶ cells per 60-mm petri dish.The following day, cells were transfected with 400 ng of L8G5-Lucreporter and 100 ng of LexA-VP16 expression vector and a combinationof empty pcineo plasmid or plasmid expressing ICP0 or partsof HDAC5 fused to GAL4 DNA-binding domain (GAL4-DB). A totalof 100 ng of a ß-galactosidase-expressing vector werealso used in each transfection. At 24 h posttransfection, cellswere washed with PBS, scraped out of the dish in 10 ml of PBS,and centrifuged at 1,000 x g for 4 min. Cell pellets were resuspendedin 200 µl of lysis buffer compatible for the luciferase-ß-galactosidasereporter gene assays (Roche), incubated at room temperaturefor 15 min, and centrifuged for 5 min at 3,000 x g. Supernatantswere then transferred in new tubes and kept on ice until use.Then, 50 µl was used for each assay, and the luciferaseactivity was measured according to the protocol provided withthe luciferase assay system kit (Promega) and normalized withrespect to the activity of ß-galactosidase measuredaccording to the protocol provided with the ß-galactosidasereporter gene assay kit (Roche).

Immunofluorescence and confocal microscopy. HeLa cells were treated according to a protocol described previously(48). The data from channels were collected separately witheightfold averaging at a resolution of 1,024 x 1,024 pixels.The microscope was a Zeiss Axioplan utilizing a x63 oil immersionobjective lens (NA 1.4). Images were acquired by using the ZeissLSM 510 META confocal microscopy software and then exportedto Adobe Photoshop for processing.

The antibodies used for immunofluorescence analyses were MAb11060 (1/1,000) and rabbit serum R190 (1/200) anti-ICP0 (20),MAb anti-ICP4 (1 µg/ml; U.S. Biologicals), MAb 12CA5 anti-HA(1 µg/ml; Boehringer Mannheim), MAb M5 anti-FLAG (1 µg/ml;Sigma), ML-19 rabbit anti-HDAC4 (1 µg/ml; Sigma), 4/21rabbit anti-SMRT (1/200; a generous gift from Hung-Ying Kao,Case Western Reserve University, Cleveland, Ohio). The secondaryantibodies used were Alexa 488-conjugated goat anti-mouse andanti-rabbit (1/200; Molecular Probes) and Cy5-conjugated goatanti-mouse and anti-rabbit (1/200; Amersham) antibodies.

RESULTS

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Results

ICP0 colocalizes with and redistributes overexpressed class I and II HDACs. Immunofluorescence experiments were performed on cotransfectedHeLa cells expressing ICP0 and an exogenous tagged version ofeither class I or class II HDACs. Overexpression of ICP0 aloneresults in the formation of ring-shaped structures in the nucleus(see Fig. 3Bi and ii, ICP0 panels). Coexpression of ICP0 withclass II HA-tagged HDAC4, -5, or -7 showed a clear redistributionof these HDACs that colocalized with ICP0 (Fig. 1Ai to iii).The same experiments were done with exogenous expressed FLAG-taggedclass I HDAC1, -2, or -3. The results obtained were slightlydifferent from those seen with class II HDACs since class IHDACs preferentially localized inside the ICP0 rings (see Fig.1Aiv, HDAC3, as an example). Hence, independent of the classthey belong to, the distribution of HDACs (either diffused orin small dots, Fig. 1A small left panels) was reorganized inthe presence of ICP0, a result not observed with other FLAG-or HA-tagged proteins (data not shown). These results, althoughobtained from HDAC overexpressing cells, at least suggesteda physical interaction between ICP0 and class I and/or classII HDACs that is strong enough to modify the distribution ofoverexpressed HDACs.

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FIG. 3. Colocalization of endogenous cellular proteins with ICP0. (A) Control of the specificity of the anti-HDAC4 ML-19 rabbit polyclonal serum by siRNA. (a) HeLa cells were transfected with three different siRNAs, ac4_334, ac4_1513, ac4_2665, targeting 5', middle, and 3' HDAC4 mRNA, respectively (for more details see Materials and Methods). After cell lysis, 10 µg of proteins was loaded for each sample, on an SDS-7.5% polyacrylamide gel and Western blots were performed with ML-19, anti-lamin A/C, and anti-actin rabbit polyclonal sera. Lamin siRNA was used as a control for specificity of the siRNA assay and for the detection of actin for protein loading. (b) HeLa cells were mock transfected (i) or transfected with siRNAac4_1513 (ii) or siRNAac4_2665 (iii) for 72 h and then treated for immunofluorescence with ML-19 to visualize the endogenous HDAC4. (B) HeLa cells were transfected with an ICP0-expressing plasmid and then analyzed at 24 h posttransfection by immunofluorescence for the detection of endogenous HDAC4 (i) and SMRT (ii) (upper right cell represents normal SMRT distribution). (C) Cells were infected for 4 h with wild-type 17+ (i) or vFXE ICP0 mutant virus (ii) or 5 h with D30EBA virus (iii and iv) at an MOI of 5 before immunofluorescence was performed. Panel iv shows a higher magnification of the region of panel iii marked by the white rectangle. ICP0, HDAC4, and SMRTwere detected by using MAb 11060, ML-19, and 4/21 rabbit anti-SMRT, respectively. The secondary antibodies used were Alexa 488-conjugated goat anti-rabbit antibody (1/200) and Cy5-conjugated goat anti-mouse immunoglobulin G (1/200). Images were collected as for Fig. 1. Bars, 5 µm.

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FIG. 1. Nuclear redistribution of ectopically expressed epitope-tagged class I or II HDACs in HeLa cells expressing ICP0. Nuclear distribution of HA epitope-tagged class II HDAC4 (i to iii), 5 (iv to vi), 7 (vii and viii), and FLAG epitope-tagged class I HDAC3 (ix and x) expressed either alone (left images) or with ICP0 (right images). ICP0 and tagged HDACs were detected by using anti-ICP0 rabbit polyclonal antibody R190 and anti-HA (12CA5) or anti-FLAG (M5) MAbs. The secondary antibodies used were Alexa 488-conjugated goat anti-mouse (1/200) and Cy5-conjugated goat anti-rabbit (1/200) antibodies. Cell samples were examined with a Zeiss LSM 510 META confocal microscope with two lasers giving excitation lines at 488 and 633 nm as described in Materials and Methods. Bars, 5 µm.

ICP0 physically interacts with class II HDACs. To check whether redistribution of exogenous HDACs by ICP0 isa consequence of their physical interaction, coimmunoprecipitationexperiments were done on cotransfected HeLa cells expressingexogenous class I or II HDACs and ICP0. ICP0 was coimmunoprecipitatedwith class II HDAC4, -5, and -7 (Fig. 2A) but not class I HDAC1,-2, and -3 (data not shown). These interactions were confirmedby GST pull-down assays by using either GST-HDACs or GST-ICP0fusion proteins (Fig. 2Cii). The noncatalytic amino-terminalregions of HDAC4 (amino acids 1 to 650), -5 (amino acids 123to 673), and -7 (amino acids 1 to 506) were sufficient to interactwith ICP0, whereas a fusion protein made of GST fused to theentire HDAC3 (amino acids 1 to 429) was unable to capture ICP0(Fig. 2B). Similarly, the amino acid region from positions 106to 241 of ICP0 contained the interaction domain with class IIHDAC4, -5, and -7, but not class I HDAC1, -2, and -3 (HDAC3is shown as an example) (Fig. 2Ci). To map the domain of interactionwithin HDAC sequences, we performed additional GST pull-downassays by using GST-HDAC5 fusion proteins. ICP0 strongly interactedwith the amino acid region from 123 to 673, and regions 123to 292 and 242 to 450 still possessed, although with a weakeraffinity, the capacity to bind ICP0 (Fig. 2D). Finally, reverseGST pull-down assays performed with in vitro-translated proteinsconfirmed that the interaction between ICP0 (Fig. 2E) and classII HDACs (Fig. 2F, HDAC7 shown as an example) are likely tobe direct. Interactions are specific for class II HDACs becausein the same experimental conditions class I HDAC1, -2, and -3never showed any binding to ICP0. This result was expected becauseICP0 interacts with the amino-terminal region of class II HDACs,which is not conserved in class I HDACs. These results showthat a class II-specific interaction between ICP0 and HDACsexists and confirm the observations made by confocal microscopy.

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FIG. 2. Physical interaction between ICP0 and class II HDACs and mapping of the interaction sites. (A) Coimmunoprecipitation of ICP0 with HA epitope-tagged class II HDAC4, -5, or -7. MAb 12CA5 was used for the immunoprecipitation (IP), and ICP0 was detected by Western blotting. Detection of the immunoprecipitated tagged-HDACs (arrowheads) was done as a control of HDAC expression. Immunoprecipitation of ICP0 with MAb 12CA5 (second upper left image) and detection of MAb 11060-immunoprecipitated ICP0 by MAb 12CA5 (first bottom left image) showed no cross-reactivity of ICP0 with this antibody. (B and Ci) GST pull-down experiments showing the capture of ICP0 (from ICP0-expressing cell extracts) (B) or tagged HDACs (from HDACs overexpressing cell extracts) (Ci) by GST-HDACs or GST-ICP0 fusion proteins, respectively. (Cii) Coomassie gel showing the expression of the GST-ICP0 fusion proteins (5 µg of proteins []) used in panel Ci. (D) Mapping of the ICP0-interacting regions in HDAC5 protein by GST pull-down with GST-HDAC5 fusion proteins. (E and F) GST pull-down experiments with in vitro-synthesized ICP0 or HA-HDAC7, respectively. ICP0 and HA- or FLAG-tagged HDACs were detected in Western blots with MAbs 11060, 12CA5, or M5, respectively.

ICP0 redistributes and colocalizes with endogenous HDAC4. To check whether ICP0 could affect the distribution of an endogenousclass II HDAC, a rabbit polyclonal serum against HDAC4 was usedto perform immunofluorescence analyses on ICP0-expressing HeLacells. The specificity of the ML-19 anti-HDAC4 rabbit serumwas checked by Western blot analyses of HeLa cell extracts transfectedwith several siRNAs targeting HDAC4 mRNA. One of three siRNAs,i.e., siRNAac4_2665, affected the signal of the HDAC4 135-kDaband detected by the serum (Fig. 3Aa). As a control, a siRNAtargeting the lamin A/C mRNA showed no decrease of HDAC4. Toconfirm the accuracy of the anti-HDAC4 serum in immunofluorescenceassays, we analyzed the endogenous HDAC4 staining in cells eithermock transfected or transfected with siRNAac4_1513 or siRNAac4_2665(Fig. 3Abi to iii). All of the data were collected by usingstrictly the same confocal parameters for the pinhole, opticalslide, detector gain, amplifier offset, amplifier gain, contrast,and brightness. The decrease of the endogenous HDAC4 signalin cells transfected with siRNAac4_2665 corresponded to theobservation made in Western blot and proved the accuracy ofthe anti-HDAC4 serum when used in immunofluorescence. Cellsshowed a relatively diffuse nuclear staining of HDAC4 (Fig.3Abi), although high magnification highlighted a dotted phenotypediffusely distributed in the whole nucleus and some weak stainingin the cytoplasm (not visible here due to contrast/brightnesseffect). Expression of ICP0 in transfected cells resulted ina change in HDAC4 distribution, which concentrated within theICP0 rings (Fig. 3Bi). To determine whether the distributionof an HDAC4-interacting protein would also be affected, we similarlydetected endogenous SMRT. As observed for HDAC4, SMRT distributionwas also modified (Fig. 3Bii, compare upper right with lowerleft nuclei). Similar results were obtained in cotransfectedcells expressing ICP0 and exogenous SMRT (data not shown). Stainingwith DAPI (4',6'-diamidino-2-phenylindole) showed no DNA inthese structures (data not shown), suggesting that ICP0 ringsare composed of several proteins probably present at a highdensity. Such proteinated structures could attract, nonspecifically,any primary or secondary antibody, which could result in falseinterpretations. We therefore intensively looked at and ruledout any kind of artifacts that could come from experimentalprocedure (i) by using different anti-mouse and anti-rabbitsecondary antibodies labeled with different fluorochromes; (ii)by using secondary antibodies highly cross-adsorbed on human,rabbit, and bovine antigens; (iii) by collecting data by sequentiallaser scanning; and finally (iv) by using Alexa 488- and Cy5-conjugatedsecondary antibodies to avoid any putative channel overlapping.In addition, the detection of several other proteins not supposedto interact with class II HDACs did not show such redistributionin ICP0-expressing cells (data not shown). These data indicatethat ICP0 modify the overall distribution of HDAC4, probablyas a result of their interaction, and suggest that other classII HDACs could behave in a similar manner. In addition, ourresults suggest that, similarly to SMRT, other class II HDAC-interactingproteins are likely to localize within the ICP0 structures.

To verify by immunofluorescence whether endogenous HDAC4 couldcolocalize with ICP0 during infection, cells were infected for4 to 5 h with wild-type virus 17+, the ICP0 mutant virus vFXE,or the replication-defective HSV-1 virus D30EBA (60) at a multiplicityof infection (MOI) of 5 (Fig. 3C). D30EBA does not express themajor viral transactivator ICP4 but overexpresses functionalICP0, which localizes 5 h postinfection in the nucleus and,unlike wild-type virus, in the cytoplasm of HeLa cells. Immunofluorescencelabeling was done as described above. As expected, most nuclearand cytoplasmic ICP0 costained with HDAC4 (Fig. 3Ci, iii, andiv). Although class II HDACs are known to shuttle from the nucleusto the cytoplasm, the cytoplasmic signal of HDAC4 is too weakcompared to the nuclear straining to be clearly visible in noninfectedHeLa cells. Therefore, the accumulation of some HDAC4 withincytoplasmic ICP0 aggregates in cells infected with D30EBA highlightsthe stability of the interaction between the two proteins (Fig.3Ciii and iv). Moreover, HDAC4, which we observed to form biggeraggregates in cells infected by vFXE, does not show a clearcolocalization with FXE (Fig. 3Cii). This is in accordance withthe GST pull-down data from Fig. 2, showing that the interactionsite of ICP0 with class II HDACs lies in the amino acid domainof ICP0(106-241) that is partly deleted in the FXE mutant protein.Finally, no degradation of endogenous HDAC4 was detected byWestern blotting, in cells infected with wild-type virus (datanot shown), which suggests that ICP0 does not interact withclass II HDACs to induce their degradation through the proteasomepathway.

ICP0 does not change the acetylation state of histone H4 and p53 proteins. Because ICP0 and TSA have similar properties in regard to reactivationof quiescent viruses, we wanted to analyze whether expressionof ICP0 could result in an overall increase of cellular acetylationactivity. The acetylation of representative histone and nonhistoneproteins, i.e., histone H4 and p53 was thus analyzed in infectedand transfected cells, respectively. Cells were infected withthe D30EBA replication-defective HSV-1 virus at an MOI of 10so that all cells were infected by at least one virus. At 24h postinfection, cells were harvested and treated to extractnuclear proteins. Unlike TSA-treated cells, infected cells didnot show any modification of the amount of acetylated histoneH4 either by immunoblotting (Fig. 4A) or by immunofluorescenceperformed at 2, 4, 6, and 24 h postinfection (data not shown).On the other hand, several nonhistone proteins, such as theproapoptotic transactivator p53, can be acetylated (30). Toanalyze whether a putative biological change could, similarlyto TSA, nonspecifically affect the acetylation of nonhistoneproteins in cells expressing ICP0, HeLa cells were cotransfectedwith plasmids expressing p53 and ICP0 or FXE, the inactive ICP0mutant protein. Total acetylated proteins were immunoprecipitatedand blotted against p53 antibodies to determine whether thelevel of acetylation of p53 changed in cells expressing ICP0.No specific increase of acetylated p53 proteins was observedin the presence of ICP0, unlike the CREB-binding protein (CBP),which is known to specifically acetylate p53 (Fig. 4B). Theslight increase of acetylated p53 in cells coexpressing ICP0correlated with an overall increase of the amount of p53 synthesized(as shown by Western blotting analysis of total cell extracts).This is most likely due to the unspecific and already describedactivation, by ICP0, of the cytomegalovirus IE promoter thatdrives the expression of the p53 ectopic gene. Taken together,these results suggest that the interaction of ICP0 with classII HDACs, both in infected and transfected cells, is unlikelyto result in general cellular changes that would modify, atleast nonspecifically and globally, the acetylation state ofhistone and nonhistone proteins. This major difference betweenICP0 and TSA underlines two potentially different mechanismsby which the drug and the viral protein would affect reactivationof quiescent viruses.

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FIG. 4. ICP0 does not modify the global acetylation activity in cells. (A) HeLa cells were infected at an MOI of 10 for 24 h by replication-defective D30EBA ICP0-overexpressing virus or treated with TSA (2 µM) for 12 h before chromatin-associated protein extraction (see Materials and Methods) and Western blotting for the detection of acetylated histone H4 (AcH4). (B) HeLa cells were transfected with indicated plasmids. At 24 h posttransfection, Western blotting was performed on cell extracts or, alternatively, acetylated proteins were immunoprecipitated with anti-acetylated lysines MAb ( ${alpha}$ -AcLys) before Western blotting. Then, 10 µg of protein was loaded for each sample on SDS-7.5% or 12.5% polyacrylamide gels for analysis. ICP0, p53, AcH4, and AcLys were detected by using MAbs 11060 and Do-7, and anti-AcH4 and anti-AcLys were detected by using rabbit polyclonal sera.

ICP0 counteracts the repressor activity of class II HDACs. In addition to their HDAC3/SMRT-associated deacetylase domain,located in the carboxy-terminal part of the protein, HDAC4,-5, and -7 possess another repressor domain in their amino-terminalregion. We thus performed GAL4-binding luciferase reporter geneassays to check whether ICP0 could affect this repressor activity(Fig. 5A). ICP0 was able to overcome, in a dose-dependent manner,the repressor activity associated to the amino-terminal partof HDAC5 (amino acids 123 to 673) (lanes 3 to 7). Conversely,ICP0 had no effect on the repression activity associated tothe carboxy-terminal region of HDAC5 (amino acids 674 to 1113,lanes 8 and 9) or to HDAC3 (lanes 10 and 11), which do not interactwith ICP0.

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FIG. 5. ICP0 counteracts the repressor activity associated with the amino-terminal region of class II HDACs. (A) HeLa cells were transfected with 400 ng of L8G5-Luc reporter and 100 ng of LexA-VP16 expression vector (46), and a combination of the indicated amount of plasmids expressing ICP0 (in nanograms) and 50 ng (+) of HDAC5 or HDAC3 proteins fused to GAL4 DNA-binding domain (GAL4-DB) (46) (see Materials and Methods). A value of 100% represents the luciferase activity calculated in the absence of HDAC5 coexpression but in the presence of 50 ng of vectors expressing the GAL4-DB; standard deviations are indicated. Lanes 2, 8, and 10 represent the controls with empty pcineo vectors. (B) HeLa cells were transfected with 1 µg of 3XMEF2-Luc reporter plasmid and as indicated (+): 200 ng of pMT2-MEF2A expressing plasmid (46) and/or plasmids expressing ICP0 (25 ng) and/or full-length or truncated HA-tagged HDAC5 (25 ng). A value of 100% represents the mean value of luciferase activity calculated in the absence of HDAC5 coexpression; standard deviations are indicated. Lanes 2, 5, 7, and 9 represent the controls with empty pcineo vectors. (C) Comparison of repression activities of truncated HDAC5(123-673), HDAC4, HDAC5, and MITR on MEF2A transcriptional activity. Experimental conditions were similar to those for panel B. A value of 100% represents the value of luciferase activity calculated in the absence of any repressor coexpression. A Western blot is shown to compare the relative amounts of each protein. (D) Same experiment as in panel C, but with the addition of 25 ng of plasmid expressing ICP0 or its RING finger mutant FXE. The black bar represents the normalized MEF2A activity in the presence of the repressor protein but in the absence of ICP0 or FXE. Western blots are shown to compare the relative amounts of each repressor protein expressed in the presence of ICP0 or FXE (lanes 1 to 4) and to show a representative amounts of ICP0 and FXE expression in all of the samples (lane 5). Western blots were performed by using 12CA5 or 11060 MAbs for the detection of HA-tagged HDACs and MITR or ICP0 and FXE, respectively. A total of 100 ng of ß-galactosidase expression vector was used in each transfection for protein amount standardization. pcineo plasmid was used in all assays to keep the amount of transfected plasmid constant as a negative control. The luciferase activity was measured 24 h posttransfection and normalized with respect to that of ß-galactosidase. All data resulted from at least three independently repeated assays; standard deviations are indicated.

The amino-terminal region of class II HDACs interacts with andinactivates MEF2 transcription factors. To confirm the resultspresented above, we investigated whether ICP0 could counterbalancethe HDAC5-induced MEF2A repression. Luciferase reporter geneassays were performed with MEF2A as an activator in the presenceof HDAC5 and/or ICP0 (Fig. 5B). As already described (46), full-lengthand amino-terminal (amino acids 1 to 673) HDAC5 strongly repressedMEF2A activity (lanes 5 and 7), unlike carboxy-terminal HDAC5(amino acids 674 to 1113) (lane 9). The addition of ICP0 hada striking effect on the MEF2A activity. First, whereas ICP0had little effect on its own on the MEF2 activated promoter(lane 3), it synergistically increased MEF2A activity in theabsence of over expressed HDAC5 (lane 4) or in the presenceof HDAC5 (amino acids 674 to 1113) (that does not bind MEF2A,lane 10). Second, ICP0 is capable to significantly release theHDAC5(123-673)-induced repression of MEF2A (compare lanes 7and 8). All of these tests were standardized with an internalß-galactosidase control to avoid variation due totransfection efficiency and the level of protein expression.This ruled out any increase of MEF2A activity due to an increaseof the protein amount when both ICP0 and MEF2A were coexpressedin the same cell. The two experiments above show that the repressoractivity associated to the amino-terminal region of a classII HDACs could be overcome by ICP0. However, in our experimentalconditions, ICP0 is unable to counterbalance the repressor activityof the HDAC5 carboxy-terminal domain that contains the deacetylase-associatedcatalytic activity (Fig. 5A, lane 9). This supports the resultsfrom Fig. 4B and confirms that rather than having a generaleffect on deacetylase-associated repressor activity, ICP0 islikely to specifically and locally (at the level of a promoter)counterbalance the repression induced by the amino-terminalregion of class II HDACs on interacting partners such as MEF2.

We then analyzed the effect of ICP0 on the repressor activityof the MEF2-interacting transcription repressor (MITR) comparedto truncated HDAC5 (amino acids 123 to 673) and full-lengthHDAC4 and HDAC5. Human MITR is a 590-amino-acid protein resultingfrom an alternative splicing of HDAC9 mRNA and shares high homologieswith the amino-terminal region of HDAC4 and -5. It interactswith MEF2 but lacks any HDAC catalytic domain (66, 81, 82).We first compared their repression effect on MEF2A transcriptionalactivity. MITR, although expressed at a lower amount (Fig. 5C,Western blot data), repressed MEF2A at a level similar to thatobserved with HDAC4 or -5 (Fig. 5C, graph data). We then performedthe same experiments in the presence of ICP0 or of its RINGfinger (amino acids 106 to 150) deletion mutant FXE. ICP0 enhancedMEF2A activity by about three- to fourfold in the presence ofMITR compared to HDAC4 and -5 (Fig. 5D, graph data). As expected,the FXE mutant, which lacks the HDAC4, -5, and -7 binding site(see Fig. 2), is unable to counterbalance the repressive effectof any of the proteins. The increase in the amount of repressorproteins present in samples expressing ICP0 compared to FXE(Fig. 5D, Western blot data) is rather in favor of an underestimationof the capacity of ICP0 to counteract their repressive effect.

These last two sets of experiments confirm that ICP0 can specificallyaffect the repression activity associated to the amino-terminalregion of class II HDACs and/or of putative short isoforms ofthese proteins. To that extent, it has been suggested that,similarly to HDAC9/MITR, shorter isoforms for other class IIHDACs are likely to exist (72, 81).

DISCUSSION

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Discussion

Viruses have evolved so that they optimally divert cellularmechanisms to their own profit. It is now clear that the acetylationpathway is one of them. Indeed, several viral proteins are acetylatedand/or recruit HATs on cellular or viral promoters and, in somecases, interfere with their enzymatic activity (reviewed inreference 10). Far fewer data are available that describe theinteraction of viral proteins with class I HDACs (13, 31, 62,79) and none with class II HDACs. The present study demonstratesfor the first time the physical and functional interactionsbetween a viral protein, HSV-1 ICP0, and several class II HDACs.

The recent description of the presence of another repressoractivity, which is not sensitive to inhibitors such as TSA,within the amino-terminal region of class II HDACs raised thequestion of the role played by these nondeacetylase domainsin the control of gene transcription. Suggestions were madeabout the need of additional mechanisms for repression by deacetylasesor about a role of the nondeacetylase domains in recruitingother corepressors to promote the formation of large enzymaticrepressor complexes. It was shown that ectopic overexpressionof HDAC4, -5, and -7 could form nuclear aggregates named matrix-associateddeacetylase (MAD) bodies (15) (see Fig. 1A, nuclear dots inHDAC4- and -5-expressing cells). Although the physiologicalsignificance of the MADs is still a matter of debate, the factis that they contain several proteins clearly involved in repressionof gene expression, such as SMRT. Our immunofluorescence dataon transfected and/or infected cells showed that endogenousand exogenous class II HDACs and SMRT undergo an ICP0-dependentredistribution. It is therefore intriguing that in transfectedcells ICP0 rings concentrate at least two of the componentsof the MADs. Given our observations of the similar accumulationof endogenous HDAC4 in ICP0 aggregates in infected cells, acompelling suggestion is that ICP0 could disrupt the activityof such protein complexes by interacting with the keystone proteinsof these structures.

Could this contribute to the general effect of ICP0 on activationof viral and cellular promoters, as described in several studies(19, 36, 37)? Our results on the lack of changes in acetylationstate of histone and nonhistone proteins in cells expressingICP0 do not support the hypothesis that ICP0-induced gene activationcould be a consequence of a global effect on acetylation. Thisfinding is in accordance with the observations that, althoughmany cellular genes are upregulated in defective or recombinantvirus- infected cells expressing ICP0, a significant proportionare concomitantly downregulated (36, 37). In addition, the lackof interaction of ICP0 with class I HDACs does not support theidea that ICP0 would enable gene expression by the sole inhibitionof deacetylation, as generally observed by the addition of TSA.

What could then be the significance of ICP0-induced overcome of class II HDAC repressor activity? As mentioned above, the amino-terminal region of class II HDAC4,-5, and -7 interacts with and represses the activity of a familyof transcription factors named MEF2 (46, 56, 75). MEF2 proteinshave initially been described as regulators of muscle development(50, 53, 57). Recently, several studies attributed to MEF2 animportant role in neuronal survival and differentiation by preventingapoptosis (27, 28, 47, 49, 58, 59). Consequently, several studieshave been done that describe the antagonist effect of classII HDACs in MEF2-regulated developmental pathways (54, 77, 78).MEF2 transcriptional activity is also controlled by class IIHDACs-related MITR, a protein sharing high homologies with theamino-terminal sequences of class II HDACs and resulting fromalternative splicing of class II HDAC9 mRNA (66, 81). It isanticipated that shorter isoforms of other class II HDACs couldalso exist (72, 81). A striking finding obtained from our genereporter assays is the efficacy of the counter effect of ICP0on the HDAC5 amino-terminal- and MITR-induced repression ofMEF2A. Interestingly, MITR transcripts were found among thedorsal root ganglia of embryonic mice, one of the sites of HSV-1latency in experimental mouse models, as well as in the brainsof adult mice (78). Because it is known that ICP0 also playsa central role in enhancing the efficiency of reactivation (33,34) and given the growing importance attributed to class IIHDACs (2, 11, 38) and MEF2 to neuronal survival, it is temptingto envisage a putative ICP0 control of the repressor activityof class II HDACs or any related proteins during reactivationfrom latency. This could boost MEF2 transcriptional activitytherefore maintaining the stressed neuron in a physiologicalstate compatible with reactivation. The results we obtainedin Fig. 5B, showing the boost of MEF2A activity in the presenceof ICP0, clearly support this hypothesis.

On the other hand, the lack of exhaustive data on the biologicalproperties of class II HDACs makes the interpretation of theirinteraction with ICP0 difficult during HSV-1 lytic infection.However, a recent study by Kao et al. (41) highlighted the criticalrole of HDAC4 in favoring double-strand break repairs in responseto DNA damage. What could then be the link with HSV-1 and ICP0?Until very recently, it was uniformly admitted that HSV-1 genomescircularized as soon as they entered the nucleus of the infectedcell. However, in a recent breakthrough study, Jackson and DeLuca(40) demonstrated that most of HSV-1 genome accumulated underlinear form during lytic infection. These authors also showedthat ICP0 is required to prevent its circularization; this isprobably due to the treatment of the ends of linear genomesas double-strand DNA breaks. It has been known for a while thatthe genomes of several DNA, as well as RNA, viruses localizeto discrete nuclear domains, called nuclear domain 10 (ND10)or PML bodies, early in infection (52). Interestingly, it hasrecently been suggested that one of the properties of ND10 couldbe to control DNA double-stranded break repair (9). ICP0 inducesboth the destabilization of ND10 and the proteasome-dependentdegradation of the catalytic subunit of the DNA-dependent proteinkinase (implicated in nonhomologous end-joining DNA repair)(51). Jackson and DeLuca (40) then proposed that this couldbe related to a property of ICP0 to participate in a generalmechanism devoted to preventing circularization of viral genomesand then to favor the onset of lytic infection. The interactionof ICP0 with at least HDAC4 and the redistribution of otherproteins implicated in DNA double-stranded break repair suchas Rad51, similarly to HDAC4 in transfected cells (P. Lomonte,unpublished observation), might reflect an additional levelat which ICP0 could control mechanisms implicated in DNA repair.

Our data thus show that class II HDACs and related proteinsmight constitute, in addition to protein degradation throughthe ubiquitin-proteasome pathway, targets for ICP0 to createa favorable environment in infected cells and reactivating neuronsthat would enable the onset of viral gene expression.

ACKNOWLEDGMENTS

Part of this study was done while P.L. was an EMBO Long-TermFellow. This study was funded by the CNRS through an ActionThématique Incitative sur Programme Jeunes Chercheurs(ATIP) to P.L., by the French Ministère de l'Educationet de la Recherche Technologique through a Programme de RechercheFondamentale en Microbiologie et Maladies Infectieuses et Parasitaires(PRFMMIP) to A.L.E. and S.D., and by L'Association pour la Recherchesur le Cancer (ARC) to A.L.E. and P.L.

We thank Eric Morency for comments on the manuscript. 11060anti-ICP0 MAb, fusion protein GST-ICP0 1-241, and several plasmidsexpressing ICP0 were provided by Roger Everett, Medical ResearchCouncil, Virology Unit, Glasgow, Scotland. ML-19 anti-HDAC4and 4/21 anti-SMRT rabbit polyclonal antibodies are generousgifts from Sigma and Hung-Ying Kao, Case Western Reserve University,Cleveland, Ohio, respectively. Plasmids expressing p53 and CBPwere provided by Pierre Hainaut, Unit of Molecular Carcinogenesis,International Agency for Research on Cancer (World Health Organization),Lyon, France, and Annick Harel-Bellan, Institut AndréLwoff, Villejuif, France, respectively. Some of the immunofluorescencedata were obtained with a confocal microscope kindly made availableby the Ecole Normale Supérieure of Lyon.

FOOTNOTES

* Corresponding author. Mailing address: Centre de Génétique Moléculaire et Cellulaire, UMR5534-CNRS, Equipe Silencing Viral et Remodelage de la Chromatine, Université Claude Bernard Lyon 1, 69622 Villeurbanne Cedex, France. Phone: (33) 4-72-4-83-79. Fax: (33) 4-72-44-05-55. E-mail: lomonte{at}cgmc.univ-lyon1.fr.

REFERENCES

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