Cellular Transcriptional Coactivator RanBP10 and Herpes Simplex Virus 1 ICP0 Interact and Synergistically Promote Viral Gene Expression and Replication

ABSTRACT

To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) regulatory protein ICP0 promotes viral gene expression and replication, we screened cells overexpressing ICP0 for ICP0-binding host cell proteins. Tandem affinity purification of transiently expressed ICP0 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP0 interacted with cell protein RanBP10, a known transcriptional coactivator, in HSV-1-infected cells. Knockdown of RanBP10 in infected HEp-2 cells resulted in a phenotype similar to that observed with the ICP0-null mutation, including reduction in viral replication and in the accumulation of viral immediate early (ICP27), early (ICP8), and late (VP16) mRNAs and proteins. In addition, RanBP10 knockdown or the ICP0-null mutation increased the level of histone H3 association with the promoters of these viral genes, which is known to repress transcription. These effects observed in wild-type HSV-1-infected HEp-2 RanBP10 knockdown cells or those observed in ICP0-null mutant virus-infected control HEp-2 cells were remarkably increased in ICP0-null mutant virus-infected HEp-2 RanBP10 knockdown cells. Our results suggested that ICP0 and RanBP10 redundantly and synergistically promoted viral gene expression by regulating chromatin remodeling of the HSV-1 genome for efficient viral replication.

IMPORTANCE Upon entry of herpesviruses into a cell, viral gene expression is restricted by heterochromatinization of the viral genome. Therefore, HSV-1 has evolved multiple mechanisms to counteract this epigenetic silencing for efficient viral gene expression and replication. HSV-1 ICP0 is one of the viral proteins involved in counteracting epigenetic silencing. Here, we identified RanBP10 as a novel cellular ICP0-binding protein and showed that RanBP10 and ICP0 appeared to act synergistically to promote viral gene expression and replication by modulating viral chromatin remodeling. Our results provide insight into the mechanisms by which HSV-1 regulates viral chromatin remodeling for efficient viral gene expression and replication.

INTRODUCTION

Herpes simplex virus 1 (HSV-1) has more than 80 different genes that fall into three major classes, designated immediate early (IE) or α, early (E) or β, and late (L) or γ, which are expressed in a regulated cascade during the HSV-1 lytic infection cycle (1). ICP0, the subject of this study, is an IE protein with a RING finger domain that confers E3 ubiquitin ligase activity, thereby mediating the ubiquitination and proteasome-dependent degradation of target proteins in HSV-1-infected cells (1–4). Based on studies using ICP0-null mutant viruses, ICP0 has been shown to be required for efficient HSV-1 gene expression and replication in cell cultures (3–7). Numerous studies of ICP0 have gradually identified the mechanisms by which ICP0 acts in HSV-1-infected cells as follows. (i) ICP0 induces the disruption of nuclear structures, designated ND10, by degrading promyelocytic leukemia protein (PML) and Sp100, major cellular components of ND10, leading to extensive redistribution of other ND10 components (4, 8). Degradation of PML and redistribution of ND10 components (e.g., ATRX and hDaxx) appear to counteract the intrinsic and interferon-mediated antiviral responses (4, 8–10). (ii) ICP0 has also been shown to degrade many other cellular proteins, including DNA-dependent protein kinase (DNA-PKcs), RNF8, RNF168, USP7, E2FBP1, IFI16, and TRIM27 (11–17). It appears that ICP0 degradation of DNA damage regulators DNA-PKcs, RNF8, and RNF168 and of IFI16, a sensor of herpesvirus DNAs, counteracts host responses activated by HSV-1 infection, including the DNA damage response and innate immune signaling, respectively (11–13, 16, 18). In addition, degradation of cellular transcription factor E2FBP1 may prevent E2FBP1 downregulation of ICP0 expression (15). Thus, ICP0 degrades cellular proteins involved in antiviral intrinsic restriction and the innate immune response. In contrast, ICP0 also appears to degrade potential positive cellular factors for HSV-1 replication, such as USP7 and TRIM27. USP7 and TRIM27 have been shown to be degraded in HSV-1-infected cells in an ICP0-dependent manner, but both proteins can promote viral replication (14, 17, 19). (iii) ICP0 has been shown to promote acetylation and eviction of histones to modulate the chromatin structure of viral genomes for efficient viral gene expression (20). In agreement with this, ICP0 has been shown to interact with and/or affect chromatin-modifying complexes; i.e., the REST/CoREST/HDAC/LSD1 complex (21, 22) and the hDaxx/ATRX complex (4, 10). In addition, the CLOCK histone acetyltransferase, which was shown to be required for efficient HSV-1 gene expression, is stabilized during HSV-1 infection and efficiently compensates for the reduction in viral growth caused by the ICP0-null mutation (23). Interestingly, ICP0 has been shown to interact with BMAL1, which binds CLOCK (24), suggesting that ICP0 may modulate CLOCK through BMAL1 to regulate acetylation of virus-associated histones. (iv) ICP0 was reported to interact with D-type cyclin cell cycle regulators and translational factor EF-1δ, and these interactions have been suggested to regulate subcellular localization of ICP0 and translation efficiency, respectively (25–27). Thus, ICP0 is a multifunctional protein that regulates a variety of cellular and viral machinery in HSV-1-infected cells.

To further define the molecular mechanism(s) by which ICP0 promotes viral gene expression and replication, in the present study we screened for host cell proteins that interact with ICP0 by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Among the putative ICP0-interacting cell proteins identified, we focused on RanBP10. RanBP10 was originally identified on the basis of its homology to RanBP9, a protein that binds a small Ras-like Ran GTPase involved in regulation of transport through nuclear pores (28, 29), and is ubiquitously expressed in human tissues and expressed in various cell types in cell cultures (28, 30). RanBP10 was reported to have multiple functions through its interaction with various proteins (30, 31). For instance, RanBP10 has been shown to regulate microtubule organization in megakaryocytes (31) and to act as a coactivator of a transcription factor androgen receptor (30). In this study, we investigated the effects of RanBP10 on HSV-1 gene expression and replication in the presence and absence of ICP0.

MATERIALS AND METHODS

Cells and viruses.

Vero, rabbit skin, HEK293T, Plat-GP, and HEp-2 cells were described previously (32–35). sh-Luc-HEp-2 cells expressing short hairpin RNA (shRNA) against firefly luciferase were described previously (36). U2OS cells (ATCC HTB-96) were purchased from the ATCC and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The HSV-1 wild-type strain HSV-1(F); recombinant virus YK711 encoding glycoprotein B (gB) fused to a Myc epitope-tobacco etch virus (TEV) protease cleavage site-Flag epitope (MEF) tag (MEF-gB); recombinant virus R7910 (ΔICP0) lacking both copies of the ICP0 gene; recombinant virus R7911 (ΔICP0-repair), in which both ICP0-null mutations were repaired; and recombinant virus YK478 encoding UL41 with an aspartic acid substitution for asparagine at amino acid 213 (UL41-D213N) were described previously (Fig. 1) (24, 32, 37, 38). R7910 (ΔICP0) and R7911 (ΔICP0-repair) were generous gifts from Bernard Roizman. R7910 (ΔICP0), R7911 (ΔICP0-repair), and YK478 (UL41-D213N) were propagated and assayed in U2OS cells. HSV-1(F) was propagated and assayed in Vero cells and, in experiments in which R7910 (ΔICP0) was used, HSV-1(F) was propagated and assayed in U2OS cells.

FIG 1.

Schematic of the genome structure of wild-type HSV-1(F) and the recombinant viruses used in this study. Line 1, wild-type HSV-1(F) genome; line 2, domain encoding the ICP0 open reading frame; lines 3 to 5 and 7; domains in recombinant virus genomes with mutations in ICP0 or UL41; line 6, domain encoding the UL41 open reading frame.

Plasmids.

pBluescript KS(+) (Stratagene) was digested with SalI, treated with T4 DNA polymerase, and religated to disrupt its SalI site, and the resulting plasmid was designated pBSΔSalI. To construct pBSΔSalI-ICP0C containing the C-terminal domain of ICP0, oligonucleotides 5′-CTAGTGCGTCGACCCGGGACGAGGGAAAACAATAAGC-3′ and 5′-GGCCGCTTATTGTTTTCCCTCGTCCCGGGTCGACGCA-3′ were annealed and cloned into the SpeI and NotI sites of pBSΔSalI. pBS-ICP0 containing the entire ICP0 open reading frame (ORF) was generated by cloning the BglII-SalI fragment of ICP0 cDNA (25) into the BamHI and SalI sites of pBSΔSalI-ICP0C. pcDNA-MEF-ICP0, an expression plasmid for ICP0 fused to the MEF tag, was constructed by cloning the EcoRI-EcoRV fragment of pBS-ICP0 into pcDNA-MEF (37). pcDNA-HA-RanBP10, an expression plasmid for RanBP10 fused to a hemagglutinin (HA) tag with an influenza virus hemagglutinin epitope, was constructed by amplifying the RanBP10 ORF by PCR from cDNA synthesized from the total RNA of HEp-2 cells using primers 5′-CGGAATTCACCATGTACCCATACGATGTTCCGGATTACGCTGGATCCACCATGGCGGCAGCGACGGCAGAC-3′ and 5′-CGGATATCCTAGTGCAAGTAGTCATCAAC-3′ and inserting the amplicon into the EcoRI and EcoRV sites of pcDNA3.1 (Invitrogen). Total RNA was isolated, and cDNAs were synthesized from the isolated RNA as described previously (36). To construct pcDNA-RanBP10, in which the HA tag in pcDNA-HA-RanBP10 was excised, pcDNA-HA-RanBP10 was digested with BamHI, treated with T4 DNA polymerase, and religated. pSSCH-RanBP10, for generating a stable cell line expressing shRNA against the 3′-untranslated region (UTR) of RanBP10 mRNA, was constructed as follows. Oligonucleotides 5′-TTTGTTACATTGGTTTATAGCATCGCTTCCTGTCACGATGCTATAAACCAATGTAACTTTTTTG-3′ and 5′-AATTCAAAAAAGTTACATTGGTTTATAGCATCGTGACAGGAAGCGATGCTATAAACCAATGTAA-3′ were annealed and cloned into BbsI and EcoRI sites of pmU6 (37). The BamHI-SalI fragment of the resulting plasmid, containing the U6 promoter and the sequence encoding shRNA against the 3′-UTR of RanBP10 mRNA, was cloned into the BamHI and SalI sites of pSSCH, which is a derivative of the retrovirus vector pMX containing a hygromycin B resistance gene (36), to produce pSSCH-RanBP10. Retrovirus vectors pMXs-HA-RanBP10 and pMXs-RanBP10, expressing HA-tagged RanBP10 and RanBP10, respectively, were constructed by cloning the EcoRI-NotI fragment of pcDNA-HA-RanBP10 and the BamHI-NotI fragment of pcDNA-RanBP10 into the EcoRI-NotI and BamHI-NotI sites of pMxs-puro (37), respectively. pMXs-Flag-RanBP10 expressing Flag-tagged RanBP10 was constructed by amplifying the RanBP10 ORF by PCR from cDNA synthesized from the total RNA of HEp-2 cells using primers 5′-CGGAATTCACCATGGACTACAAAGACGATGACGACAAGGCGGCAGCGACGGCAGACCC-3′ and 5′-CCGCTCGAGCTAGTGCAAGTAGTCATCAAC-3′ and inserting the amplicon into the EcoRI and XhoI sites of pMXs-puro.

Identification of proteins that interact with ICP0.

HEK293T cells were transfected with pcDNA-MEF-ICP0 using polyethylenimine as described previously (39), harvested at 36 h posttransfection, and lysed in 0.1% NP-40 buffer (50 mM Tris-HCl [pH 8.0], 120 mM NaCl, 50 mM NaF, 0.1% NP-40) containing protease and phosphatase inhibitor cocktails (Nacalai Tesque). After centrifugation, the supernatants were immunoprecipitated with an anti-Myc monoclonal antibody, and the immunoprecipitates were incubated with AcTEV protease (Invitrogen) for 1 h at room temperature. After another centrifugation, the supernatants were immunoprecipitated with an anti-Flag M2 affinity gel (Sigma), and the immunoprecipitates were washed three times with 0.1% NP-40 buffer and two times with wash buffer (50 mM Tris-HCl [pH 8.0], 120 mM NaCl, 50 mM NaF). Flag elution buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mg Flag peptide/ml) was added, and the immunoprecipitates were rotated for 2 h at 4°C. The eluted protein solution was treated with trypsin and analyzed by nano-liquid chromatography–tandem mass spectrometry (nanoLC-MS/MS) as described previously (40). For this analysis, we used Q-STAR Elite (AB SCIEX) coupled with Dina (KYA Technologies). The MS/MS signals were then analyzed against the human proteins in the RefSeq database (National Center for Biotechnology Information; 35,853 protein sequences as of 4 February 2013) using the Mascot algorithm (version 2.4.1; Matrix Science) with the following parameters: variable modifications, methionine oxidation, protein N-terminal acetylation, and pyro-glutamination for N-terminal glutamine; maximum missed cleavages, 2; peptide mass tolerance, 200 ppm; and MS/MS tolerance, 0.5 Da. Protein identification was based on the criterion of having at least one MS/MS data signal with a Mascot score that exceeded the threshold (P < 0.05).

Mutagenesis of HSV-1 genomes and generation of recombinant HSV-1.

To generate recombinant virus YK322 in which the two ICP0 copies were each fused to an MEF tag (MEF-ICP0), a two-step Red-mediated mutagenesis procedure was carried out using Escherichia coli GS1783 containing pYEbac102 (32), a full-length infectious HSV-1(F) clone, as described previously (41), except with primers 5′-CGACCCCCAGGGACCCTCCGTCCGCGACCCTCCAACCGCATACGACCCCCATGGAGCAAAAGCTCATTTC-3′ and 5′-TGGGGGCGGCCCTCAGGCCGGCGGGTACTCGCTCCGGGGCGGGGCTCCATATCTTTGTCATCGTCGTCCT-3′ (Fig. 1). Plaques of progeny viruses from the transfection were isolated three times and analyzed by PCR using primers 5′-TCTCCGCATCACCACAGAAG-3′ and 5′-GACCACCATGACGACGACTC-3′ to confirm that both copies of the ICP0 gene were tagged with MEF.

Antibodies.

The commercial antibodies used in this study were mouse monoclonal antibodies to Flag (M2; Sigma), Myc (PL14; MBL), HA (TANA2; MBL), β-actin (AC15; Sigma), ICP0 (1112; Goodwin Institute), ICP4 (58S; ATCC), ICP27 (8.F.137B; Abcam), and ICP8 (10A3 [Millipore] and HB-8180 [ATCC]); and rabbit polyclonal antibodies to RanBP10 (ab150930; Abcam), Flag (PM020; MBL), HSV-1 (B0114; DakoCytomation), and VP16 (CAC-CT-HSV-UL48; CosmoBio). The two mouse monoclonal antibodies against ICP8 (10A3 and HB-8180) were used for immunoblotting and immunofluorescence, respectively. Mouse polyclonal antibodies to the C-terminal domain of ICP22 (42) and rabbit polyclonal antibodies to the N-terminal domain of ICP0 (25) were described previously.

Antibody analysis.

Immunoprecipitation, immunoblotting, and immunofluorescence were carried out as described previously (25, 43). The amount of protein in immunoblot bands was quantitated using the ImageQuant LAS 4000 system with ImageQuant TL7.0 analysis software (GE Healthcare Life Sciences).

Establishment of cell lines stably expressing RanBP10 or shRNA against RanBP10.

Recombinant retroviruses for these studies were generated as described previously (34). HEp-2 cells expressing HA-tagged RanBP-10 (HEp-2/HA-RanBP10 cells), Flag-tagged RanBP10 (HEp-2/Flag-RanBP10 cells), or shRNA against RanBP10 (sh-RanBP10-HEp-2 cells) were isolated from HEp-2 cells infected with retrovirus-containing supernatants of Plat-GP cells that had been transfected with pMXs-HA-RanBP10, pMXs-Flag-RanBP10, or pSSCH-RanBP10, respectively, and selected with 1 μg puromycin/ml (HEp-2/HA-RanBP10 and HEp-2/Flag-RanBP10 cells) or with 50 μg hygromycin B/ml (sh-RanBP10-HEp-2 cells).

Establishment of sh-RanBP10-HEp-2 cells exogenously expressing RanBP10.

sh-RanBP10-HEp-2 cells were transduced by infection with retrovirus-containing supernatants of Plat-GP cells that had been transfected with pMXs-RanBP10 and selected with 1 μg puromycin/ml maintenance medium, which led to the isolation of sh-RanBP10/RanBP10-HEp-2 cells.

Assay for cell viability.

The viability of sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells was assayed using a cell counting kit-8 (Dojindo) according to the manufacturer's instructions.

Determination of plaque size.

sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells were infected with wild-type HSV-1(F), R7910 (ΔICP0), or R7911 (ΔICP0-repair) under plaque assay conditions, and plaques produced by each of the viruses were assayed as described preciously (44) except that plaques were visualized by immunofluorescence using the rabbit polyclonal antibody against HSV-1.

Quantitative reverse transcription PCR (qPCR).

Total RNA was isolated from infected cells with a SuperPrep cell lysis kit for qPCR (Toyobo) according to the manufacturer's instructions, and cDNA was synthesized from the isolated RNA as described above. The amount of cDNA of specific genes was quantitated using the Universal ProbeLibrary (Roche) with TaqMan Master (Roche) and the LightCycler 1.1 system (Roche) according to the manufacturer's instructions. Gene-specific primers and universal probes were designed using ProbeFinder software (Roche). The primer sequences and probes for ICP27 were 5′-TCCGACAGCGATCTGGAC-3′, 5′-TCCGACGAGGAACACTCC-3′, and Universal ProbeLibrary probe 56; those for ICP8 were 5′-ACAGCTGCAGATCGAGGACT-3′, 5′-CCATCATCTCCTCGCTTAGG-3′, and Universal ProbeLibrary probe 65; those for VP16 were 5′-GCGCTCTCTCGTTTCTTCC-3′, 5′-GGCCAACACGGTTCGATA-3′, and Universal ProbeLibrary probe 52; and those for 18S rRNA were 5′-GCAATTATTCCCCATGAACG-3′, 5′-GGGACTTAATCAACGCAAGC-3′, and Universal ProbeLibrary probe 48. The amount of ICP27, ICP8, and VP16 mRNA expression was normalized to the amount of 18S rRNA expression. The relative amount of each gene expression was calculated using the comparative cycle threshold (2^−ΔΔCT) method (45).

ChIP and real-time PCR.

sh-RanBP10-HEp-2 and sh-RanBP10/RanBP10-HEp-2 cells (5 × 10⁶) were grown in 100-mm dishes and infected with R7910 (ΔICP0) or R7911 (ΔICP0-repair) at a multiplicity of infection (MOI) of 5. At 18 h postinfection, cells were fixed with 1% formaldehyde for 10 min. The formaldehyde was quenched by the addition of glycine to a final concentration of 125 mM for 5 min. Cells were washed with phosphate-buffered saline (PBS) three times, resuspended in lysis buffer 1 (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), and incubated for 10 min on ice. Nuclei were pelleted by microcentrifugation, resuspended in lysis buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl [pH 8.0]), and incubated for 10 min on ice. After centrifugation, pellets were resuspended in lysis buffer 3 (100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl [pH 8.0], 0.1% sodium deoxycholate, 0.5% sodium N-lauroylsarcosinate) and sonicated 10 times with 30-s pulses using a sonicator (UR-21P; TOMY) to produce DNA fragments of ∼200 to ∼1,000 bp in length. Triton X-100 was added to the samples to a final concentration of 1%, and the samples were then clarified by microcentrifugation at 15,000 × g for 10 min at 4°C. The supernatants were precleared by rotation with Dynabeads protein G (Novex) for 30 min at 4°C, and 10% of the precleared samples were removed and used as input samples. For immunoprecipitation samples, 3 μg anti-histone H3 antibody (ab1791; Abcam) or normal rabbit IgG (MBL) was preincubated with Dynabeads protein G in chromatin immunoprecipitation (ChIP) dilution buffer (0.5% bovine serum albumin [BSA] in PBS) overnight at 4°C. Immunoprecipitation with antibody bound to Dynabeads protein G was carried out at 4°C for 4 h. The beads were washed six times with radioimmunoprecipitation assay (RIPA) buffer (50 mM HEPES-KOH [pH 7.5], 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate), and once with TNE buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 50 mM NaCl). DNA-protein complexes were eluted from antibody on the beads by addition of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS), followed by incubation for 15 min at 65°C. Immunoprecipitate and input samples were reverse cross-linked overnight at 65°C and then incubated for 2 h at 37°C with RNase A (Sigma) to a final concentration of 0.2 μg/μl. Proteinase K and CaCl₂ were added to the samples to final concentrations of 0.2 μg/μl and 5.25 mM, respectively, and allowed to react at 55°C for 1 h. DNA was purified using a NucleoSpin gel and PCR clean-up kit (Machery-Nagel). The amount of specific genes was quantitated using 2.5 μl DNA and 1 μM each primer with 2× Sybr green mix (Roche) and the LightCycler 1.1 system (Roche) according to the manufacturer's instructions. Primer sequences for the ICP27 promoter were 5′-CACCACCAGAGGCCATATCCGACA-3′ and 5′-AGCATATCAATGTCAGTCGCCATGACCG-3′, for the ICP8 promoter were 5′-GTCCTTCTGTCAATCGGTCC-3′ and 5′-GATTTTGACGCTCGGGAGAC-3′, and for the VP16 promoter were 5′-GCCGCCCCGTACCTCGTGAC-3′ and 5′-CAGCCCGCTCCGCTTCTCG-3′.

RESULTS

Identification of cell proteins that interact with ICP0.

To identify host cell proteins that interact with ICP0, we used a tandem affinity purification approach coupled with mass spectrometry-based proteomics technology. These experiments identified 80 cell proteins that coimmunoprecipitated with transiently expressed MEF-tagged ICP0 (data not shown). The proteins identified included USP7, which has previously been shown to bind ICP0 (17, 46). Of the putative ICP0-interacting cell proteins identified, we focused on RanBP10. To verify the interaction between ICP0 and RanBP10, HEK293T cells were transfected with a plasmid expressing MEF-tagged ICP0 in combination with either empty vector pcDNA-MEF, another empty vector, pcDNA3.1, or a plasmid expressing HA-tagged RanBP10, lysed, immunoprecipitated with anti-Flag or anti-HA antibody, and immunoblotted with anti-HA or anti-Flag antibody, respectively. When HA-tagged RanBP10 and MEF-tagged ICP0 were coexpressed, anti-Flag antibody coprecipitated HA-tagged RanBP10 and MEF-tagged ICP0 (Fig. 2A). In contrast, when only HA-tagged RanBP10 was expressed, HA-tagged RanBP10 was not coprecipitated by anti-Flag antibody (Fig. 2A). In a reciprocal experiment, when HA-tagged RanBP10 and MEF-tagged ICP0 were coexpressed, anti-HA antibody coprecipitated MEF-tagged ICP0 and HA-tagged RanBP10 (Fig. 2B), but when only MEF-tagged ICP0 was expressed, MEF-tagged ICP0 was not coprecipitated by anti-HA antibody (Fig. 2A). Thus, the interaction of ICP0 and RanBP10 was confirmed in cells in which both were ectopically expressed.

FIG 2.

Interaction of ICP0 with RanBP10. HEK293T cells were transfected with pcDNA-MEF-ICP0 (A and B) in combination with either pcDNA-MEF (A), pcDNA3.1 (B), or pcDNA-HA-RanBP10 (A and B). At 36 h posttransfection, the cells were harvested, immunoprecipitated (IP) with anti-Flag (A) or anti-HA (B) antibody, and analyzed by immunoblotting (IB) with the indicated antibodies. WCE, whole-cell extract; α, anti-.

Characterization of the recombinant viruses and cell lines generated in this study.

To facilitate studies of the interaction between ICP0 and RanBP10 in HSV-1-infected cells, we constructed recombinant HSV-1 YK322 (MEF-ICP0) in which the N terminus of ICP0 was tagged with MEF (Fig. 1) and HEp-2 cell lines expressing HA-tagged RanBP10 (HEp-2/HA-RanBP10) and Flag-tagged RanBP10 (HEp-2/Flag-RanBP10 cells). As shown in Fig. 3A, Vero and HEp-2 cells infected with YK322 (MEF-ICP0) expressed MEF-tagged ICP0 and produced ICP0 and ICP27 proteins at levels similar to those produced by cells infected with wild-type HSV-1(F) (Fig. 3A). The growth curves of YK322 (MEF-ICP0) in Vero (Fig. 3B and C) and HEp-2 (Fig. 3D and E) cells infected at MOIs of 0.01 and 5 were similar to those of wild-type HSV-1(F) (Fig. 3B to E). HEp-2/HA-RanBP10 and HEp-2/Flag-RanBP10 cells efficiently expressed HA-tagged RanBP10 (Fig. 4A) and Flag-tagged RanBP10 (Fig. 5A), respectively, with and without HSV-1 infection.

FIG 3.

Characterization of the recombinant viruses generated in this study. (A) Vero and HEp-2 cells were mock infected or infected with wild-type HSV-1(F) or YK322 (MEF-ICP0) at an MOI of 5 for 18 h and then analyzed by immunoblotting with the indicated antibodies. (B to E) Vero (B and C) and (D and E) HEp-2 cells were infected with wild-type HSV-1(F) or YK322 (MEF-ICP0) at an MOI of 5 (B and D) or 0.01 (C and E). Total virus from the cell culture supernatants and infected cells was harvested at the indicated times and assayed on Vero cells. α, anti-.

FIG 4.

Interaction of ICP0 with RanBP10 in HSV-1-infected cells. (A) HEp-2/HA-RanBP10 and HEp-2 cells were mock infected or infected with wild-type HSV-1(F) at an MOI of 5 for 8 h and then analyzed by immunoblotting with the indicated antibodies. (B) HEp-2 and HEp-2/HA-RanBP10 cells were infected with wild-type HSV-1(F) at an MOI of 5 for 8 h and then harvested, immunoprecipitated (IP) with anti-HA antibody, and analyzed by immunoblotting (IB) with the indicated antibodies. WCE, whole-cell extract. (C) HEp-2/HA-RanBP10 cells were infected with wild-type HSV-1(F), YK322 (MEF-ICP0), or YK711 (MEF-gB) at an MOI of 5 for 8 h and then harvested, immunoprecipitated (IP) with anti-Myc antibody, and analyzed by immunoblotting (IB) with the indicated antibodies. WCE, whole-cell extract. (D) HEp-2 and HEp-2/HA-RanBP10 cells were mock infected or infected with wild-type HSV-1(F) at an MOI of 5 for 8 h and then fixed, permeabilized, stained with anti-HA and anti-ICP0 antibodies, and examined by confocal microscopy. α, anti.

FIG 5.

Localization of RanBP10 and ICP8 in HSV-1-infected cells. (A) HEp-2/Flag-RanBP10 and HEp-2 cells were mock infected or infected with wild-type HSV-1(F) at an MOI of 5 for 18 h and then analyzed by immunoblotting with the indicated antibodies. (B) HEp-2/Flag-RanBP10 and HEp-2 cells were mock infected or infected with wild-type HSV-1(F) at an MOI of 5 for 18 h and then fixed, permeabilized, stained with anti-Flag and anti-ICP8 antibodies, and examined by confocal microscopy. α, anti.

Interaction of ICP0 with RanBP10 in HSV-1-infected cells.

To examine whether ICP0 interacted with RanBP10 in HSV-1-infected cells, three series of experiments were carried out. In the first series of experiments, HEp-2 and HEp-2/HA-RanBP10 cells were infected with wild-type HSV-1(F) at an MOI of 5 for 8 h and then lysed, immunoprecipitated with anti-HA antibody, and immunoblotted with anti-ICP0 and anti-HA antibodies. As shown in Fig. 4B, anti-HA antibody coprecipitated ICP0 and HA-tagged RanBP10 from lysates of wild-type HSV-1(F)-infected HEp-2/HA-RanBP10 cells but not from lysates of HSV-1(F)-infected HEp-2 cells. In the second series of experiments, HEp-2/HA-RanBP10 cells were infected with wild-type HSV-1(F), YK322 (MEF-ICP0), or YK771 (MEF-gB) at an MOI of 5 for 8 h and then lysed, immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-HA and anti-Myc antibodies. As shown in Fig. 4C, anti-Myc antibody coprecipitated HA-tagged RanBP10 and MEF-tagged ICP0 from lysates of HEp-2/HA-RanBP10 cells infected with YK322 (MEF-ICP0) but not from lysates of HEp-2/HA-RanBP10 cells infected with wild-type HSV-1(F) or YK771 (MEF-gB). In the third series of experiments, HEp-2 and HEp-2/HA-RanBP10 cells infected with wild-type HSV-1(F) at an MOI of 5 for 8 h were fixed and analyzed by immunofluorescence with anti-ICP0 and anti-HA antibodies. As shown in Fig. 4D, in mock-infected HEp-2/HA-RanBP10 cells, HA-tagged RanBP10 was detected diffused in the cytoplasm and as punctate dots in the nucleus. In contrast, in wild-type HSV-1(F)-infected HEp-2/HA-RanBP10 cells, HA-tagged RanBP10 was mainly detected and colocalized with ICP0 in discrete domains in the nucleus (Fig. 4D).

Collectively, these experiments indicated that ICP0 interacted with RanBP10 in HSV-1-infected cells.

Colocalization of RanBP10 and ICP8 in HSV-1-infected cells.

To further investigate the localization of RanBP10 in HSV-1-infected cells, HEp-2/Flag-RanBP10 and HEp-2 cells were mock infected or infected with wild-type HSV-1(F) at an MOI of 5 for 18 h and then fixed and analyzed by immunofluorescence with anti-ICP8 and anti-Flag antibodies. ICP8 is a marker for intracellular replication compartments where HSV-1 DNA replication and transcription take place (47). As shown in Fig. 5B, Flag-tagged RanBP10 colocalized with ICP8 in the nuclei of HSV-1(F)-infected HEp-2/Flag-RanBP10 cells. This result suggested that RanBP10 was recruited to the HSV-1 replication compartments in infected cells.

Effect of RanBP10 knockdown on HSV-1 replication in the presence and absence of ICP0.

To investigate the role(s) of RanBP10 in HSV-1 replication in the presence and absence of ICP0, we generated HEp-2 cell lines stably expressing shRNA against the 3′-UTR of RanBP10 mRNA (sh-RanBP10-HEp-2) to knock down RanBP10 expression. In addition, to examine whether the phenotype(s) observed in sh-RanBP10-HEp-2 cells was due to a nonspecific effect(s) of the shRNA, we generated sh-RanBP10/RanBP10-HEp-2 cells in which RanBP10 was expressed exogenously by transduction of sh-RanBP10-HEp-2 cells with a retrovirus vector expressing RanBP10. In the following experiments, we used sh-Luc-HEp-2 cells expressing shRNA against firefly luciferase mRNA (36) as a control cell line. As shown in Fig. 6A, RanBP10 expression in sh-RanBP10-HEp-2 cells was less than that in both sh-Luc-HEp-2 and sh-RanBP10/RanBP10-HEp-2 cells. We noted that RanBP10 expression in sh-RanBP10/RanBP10-HEp-2 cells was greater than that in sh-Luc-HEp2 cells (Fig. 6A). In contrast, the viability of sh-Luc-HEp2 cells was similar to that of sh-RanBP10-HEp-2 and sh-RanBP10/RanBP10-HEp-2 cells (Fig. 6B), indicating that the level of RanBP10 expression had no effect on HEp-2 cell viability.

FIG 6.

Characterization of sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells. (A) RanBP10 expression in sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells was analyzed by immunoblotting with the indicated antibodies. (B) The cell viability of sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells was assayed 24 h after 5 × 10³ cells were seeded on 96-well plates. Each value is the mean ± standard error of the results of triplicate experiments and is expressed relative to the mean for sh-Luc-HEp-2 cells, which was normalized to 100%.

sh-Luc-HEp-2 and sh-RanBP10-HEp-2 cells were then infected with wild-type HSV-1(F), R7910 (ΔICP0), or R7911 (ΔICP0-repair) at an MOI of 0.01, and progeny virus titers were assayed at various times postinfection (Fig. 7). In addition, sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells were infected with wild-type HSV-1(F), R7910 (ΔICP0), R7911 (ΔICP0-repair), or YK478 (UL41-D213N) at an MOI of 0.01, and progeny virus titers were assayed at 48 h postinfection. In agreement with previous reports (5–7), the ICP0-null mutation reduced virus growth in control sh-Luc-HEp-2 cells: the progeny virus titer of R7910 (ΔICP0) was 15-fold less than that of wild-type HSV-1(F) in sh-Luc-HEp-2 cells at 48 h postinfection (Fig. 7 and 8A). Similarly, the progeny virus titer of YK478 (UL41-D213N) was 11-fold less than that of wild-type HSV-1(F) in sh-Luc-HEp2 cells (Fig. 8A). We used YK478 (UL41-D213N) to examine the specific effect of the ICP0-null mutation on viral replication in combination with RanBP10 knockdown.

FIG 7.

Effect of RanBP10 on HSV-1 replication. (A) sh-Luc-HEp-2 and (B) sh-RanBP10-HEp-2 cells were infected with wild-type HSV-1(F), R7910 (ΔICP0), or R7911 (ΔICP0-repair) at an MOI of 0.01. Total virus from the cell culture supernatants and infected cells was harvested at the indicated times and assayed on U2OS cells.

FIG 8.

Effect of RanBP10 and/or ICP0 on progeny virus titers. (A) sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells were infected with wild-type HSV-1(F), R7910 (ΔICP0), R7911 (ΔICP0-repair), or YK478 (UL41-D213N) at an MOI of 0.01. Total virus from cell culture supernatants and infected cells was harvested at 48 h postinfection and assayed on U2OS cells. (B) Fold reduction in virus yield from panel A of the indicated viruses in sh-RanBP10-HEp-2 cells relative to that in sh-Luc-HEp-2 cells. (C) Fold reduction in virus yield from panel A of the indicated viruses in sh-RanBP10-HEp-2 cells relative to that in sh-RanBP10/RanBP10-HEp-2 cells. (D) Fold reduction in virus yield from panel A of R7910 (ΔICP0) relative to that of wild-type HSV-1(F) in the indicated cells, of R7910 (ΔICP0) relative to that of R7911 (ΔICP0-repair) in the indicated cells, and of YK478 (UL41-D213N) relative to that of wild-type HSV-1(F) in the indicated cells. Each value is the mean ± standard error of data from three independent experiments. Asterisks indicate significant differences (*, P < 0.0001; **, P < 0.001; ***, P < 0.01; ****, P < 0.05) by analysis of variance (ANOVA) and Tukey's test.

As shown in Fig. 7, growth of wild-type HSV-1(F) in sh-RanBP10-HEp-2 cells was less than that in sh-Luc-HEp-2 cells. Interestingly, RanBP10 knockdown reduced replication of R7910 (ΔICP0) much more than replication of wild-type HSV-1(F), R7911 (ΔICP0-repair), and YK478 (UL41-D213N) (Fig. 7 and 8A to C). Thus, the progeny virus titer of R7910 (ΔICP0) in sh-RanBP10-HEp-2 cells at 48 h postinfection was 360-fold lower than that in sh-Luc-HEp-2 cells (Fig. 8A and B). In contrast, the progeny virus titers of wild-type HSV-1(F), R7911 (ΔICP0-repair), and YK478 (UL41-D213N) in sh-RanBP10-HEp-2 cells at 48 h postinfection were only 7.1-, 9.5- and 15-fold lower than those in sh-Luc-HEp-2 cells, respectively (Fig. 8A and B). Similar results were also obtained with sh-RanBP10-HEp2 and sh-RanBP10/RanBP10-HEp2 cells infected with wild-type HSV-1(F), R7910 (ΔICP0), R7911 (ΔICP0-repair), or YK478 (UL41-D213N) (Fig. 8A and C). In addition, the ICP0-null mutation reduced viral replication in sh-RanBP10-HEp-2 cells much more than in sh-Luc-HEp-2 cells and in sh-RanBP10/RanBP10-HEp-2 cells (Fig. 8A and D). The progeny virus titer of R7910 (ΔICP0) in sh-RanBP10-HEp-2 cells at 48 h postinfection was 792- and 137-fold lower than that of wild-type HSV-1(F) and R7911 (ΔICP0-repair) in sh-RanBP10-HEp-2 cells (Fig. 8A and D), whereas the progeny virus titer of R7910 (ΔICP0) in sh-Luc-HEp-2 cells was only 15- and 3.7-fold lower than that of wild-type HSV-1(F) and R7911 (ΔICP0-repair) in sh-Luc-HEp-2 cells (Fig. 8A and D). In contrast, the progeny virus titers of YK478 (UL41-D213N) in sh-RanBP10-HEp-2 and sh-Luc-HEp-2 cells at 48 h postinfection were only 29- and 11-fold lower than those of wild-type HSV-1(F) in sh-RanBP10-HEp-2 and sh-Luc-HEp-2 cells, respectively (Fig. 8A and D). Similar results were also obtained with sh-RanBP10-HEp2 and sh-RanBP10/RanBP10-HEp2-cells infected with wild-type HSV-1(F), R7910 (ΔICP0), R7911 (ΔICP0-repair), or YK478 (UL41-D213N) (Fig. 8A and D).

We also analyzed plaque sizes in sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells infected with either wild-type HSV-1(F), R7910 (ΔICP0), or R7911 (ΔICP0-repair) to examine the significance of RanBP10 for viral cell-cell spread in the presence and absence of ICP0. In agreement with the growth properties of these viruses shown above (Fig. 7 and 8), plaques produced in sh-RanBP10-HEP-2 cells by wild-type HSV-1(F) or R7911 (ΔICP0-repair) were smaller than those produced by wild-type HSV-1(F) or R7911 (ΔICP0-repair) in sh-Luc-HEp-2 and sh-RanBP10/RanBP10-Hep-2 cells (Fig. 9A). In addition, RanBP10 knockdown reduced the plaque sizes in R7910 (ΔICP0)-infected cells more than in wild-type HSV-1(F)- and R7911 (ΔICP0-repair)-infected cells (Fig. 9A to C), and the ICP0-null mutation reduced the plaque sizes in sh-RanBP10-HEp-2 cells more than in sh-Luc-HEp-2 cells and sh-RanBP10/RanBP10-HEp-2 cells (Fig. 9A, D, and E).

FIG 9.

Effect of RanBP10 on virus plaque size. sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells in 24-well plates were infected with 100 PFU of wild-type HSV-1(F), R7910 (ΔICP0), or R7911 (ΔICP0-repair) under plaque assay conditions. The diameters of 10 single plaques for each of the indicated viruses were measured 48 h postinfection. (B) Fold reduction in the plaque size in the experiment in panel A in sh-RanBP10-HEp-2 cells infected by the indicated viruses relative to that in sh-Luc-HEp-2 cells infected by the same viruses. (C) Fold reduction in the plaque size in the experiment in panel A in sh-RanBP10-HEp-2 cells infected by the indicated viruses relative to that in sh-RanBP10/RanBP10-HEp-2 infected by the same viruses. (D) Fold reduction in the plaque size in the experiment in panel A in the indicated cells infected by R7910 (ΔICP0) relative to that in the same cells infected by wild-type HSV-1(F). (E) Fold reduction in the plaque size in the experiment in panel A in the indicated cells infected by R7910 (ΔICP0) relative to that in the same cells infected by R7911 (ΔICP0-repair). Each value is the mean ± standard error of the measured plaque sizes and is representative of three independent experiments. Asterisks indicate significant differences (*, P < 0.0001) by ANOVA and Tukey's test.

These results indicate that RanBP10 was required for efficient HSV-1 replication and cell-cell spread and that although both RanBP10 and ICP0 were needed for efficient HSV-1 replication and cell-cell spread, each was able to partially compensate for the absence of the other.

Effect of RanBP10 knockdown on HSV-1 ICP27, ICP8, and VP16 expression in the presence and absence of ICP0.

We next investigated the effect of RanBP10 on expression of HSV-1 genes in the presence and absence of ICP0. sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells were infected with wild-type HSV-1(F), R7910 (ΔICP0), or R7911 (ΔICP0-repair) at an MOI of 5, and at 18 h postinfection, the levels of mRNA and protein expression of IE gene ICP27, E gene ICP8, and L gene VP16 were analyzed. As shown in Fig. 10, 11A, and 12A, sh-RanBP10-HEp-2 cells infected with wild-type HSV-1(F) or R7911 (ΔICP0-repair) produced less ICP27, ICP8, and VP16 mRNAs and proteins than sh-Luc-HEp-2 and sh-RanBP10/RanBP10-HEp-2 cells infected with wild-type HSV-1(F) or R7911 (ΔICP0-repair). In agreement with the effects of RanBP10 knockdown and the ICP0-null mutation on viral replication described above (Fig. 7 and 8), RanBP10 knockdown reduced the accumulation of ICP27, ICP8, and VP16 mRNAs and proteins in R7910 (ΔICP0)-infected cells more than in wild-type HSV-1(F)- and R7911 (ΔICP0-repair)-infected cells (Fig. 10, 11A to C, and 12A to C). In addition, the ICP0-null mutation reduced the accumulation of ICP27, ICP8, and VP16 mRNAs and proteins in sh-RanBP10-HEp-2 cells more than in sh-Luc-HEp-2 cells and sh-RanBP10/RanBP10-HEp-2 cells (Fig. 10, Fig. 11A, D, and E, and Fig. 12A, D, and E). These results indicated that, in addition to ICP0, RanBP10 was also required for efficient expression of ICP27, ICP8, and VP16 and that both RanBP10 and ICP0 were able to partially compensate for the absence of the other.

FIG 10.

Effect of RanBP10 on expression of HSV-1 proteins. (A) sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells were infected with wild-type HSV-1(F), R7910 (ΔICP0), or R7911 (ΔICP0-repair) at an MOI of 5 for 18 h and then harvested and analyzed by immunoblotting with the indicated antibodies. Data are representative of three independent experiments.

FIG 11.

Effect of RanBP10 and/or the ICP0 on expression of HSV-1 proteins. (A) The amounts of ICP27, ICP8, and VP16 proteins from the experiment in Fig. 10 were quantitated and normalized to those of β-actin protein. (B) Fold reduction in the amount of viral protein in the experiment in panel A in sh-RanBP10-HEp-2 cells infected by the indicated viruses relative to that in sh-Luc-HEp-2 cells infected by the same viruses. (C) Fold reduction in the amount of viral protein in the experiment in panel A in sh-RanBP10-HEp-2 cells infected by the indicated viruses relative to that in sh-RanBP10/RanBP10-HEp-2 infected by the same viruses. (D) Fold reduction in the amount of viral protein in the experiment in panel A in the indicated cells infected by R7910 (ΔICP0) relative to that in the same cells infected by wild-type HSV-1(F). (E) Fold reduction in the amount of viral protein in the experiment in panel A in the indicated cells infected by R7910 (ΔICP0) relative to that in the same cells infected by R7911 (ΔICP0-repair). Each value is the mean ± standard error of data from three independent experiments. Asterisks indicate significant differences (*, P < 0.0001; ***, P < 0.01) by ANOVA and Tukey's test.

FIG 12.

Effect of RanBP10 and/or ICP0 on expression of HSV-1 mRNAs. (A) sh-Luc-HEp-2, sh-RanBP10-HEp-2, and sh-RanBP10/RanBP10-HEp-2 cells were infected with wild-type HSV-1(F), R7910 (ΔICP0), or R7911 (ΔICP0-repair) at an MOI of 5 for 18 h and then harvested and analyzed for the amounts of ICP27, ICP8, and VP16 mRNA by quantitative RT-PCR. (B) Fold reduction in the amount of viral mRNAs in the experiment in panel A in sh-RanBP10-HEp-2 cells infected by the indicated viruses relative to that in sh-Luc-HEp-2 cells infected by the same viruses. (C) Fold reduction in the amount of viral mRNAs in the experiment in panel A in sh-RanBP10-HEp-2 cells infected by the indicated viruses relative to that in sh-RanBP10/RanBP10-HEp-2 infected by the same viruses. (D) Fold reduction in the amount of viral mRNAs in the experiment in panel A in the indicated cells infected by R7910 (ΔICP0) relative to that in the same cells infected by wild-type HSV-1(F). (E) Fold reduction in the amount of viral mRNAs in the experiment in panel A in the indicated cells infected by R7910 (ΔICP0) relative to that in the same cells infected by R7911 (ΔICP0-repair). Each value is the mean ± standard error of data from three independent experiments. Asterisks indicate significant differences (*, P < 0.0001; **, P < 0.001; ***, P < 0.01; ****, P < 0.05) by ANOVA and Tukey's test.

Effect of RanBP10 on the association of ICP27, ICP8, and VP16 promoters with histone H3 in the presence and absence of ICP0.

It is known that HSV-1 gene expression, like eukaryotic gene expression, is regulated by changes in chromatin structure and that viral gene expression is regulated by the association of histones with the viral genome (48, 49). To investigate the effect of RanBP10 on chromatin association with the HSV-1 genome in the presence and absence of ICP0, we carried out ChIP assays of sh-RanBP10/RanBP10-HEp-2 and sh-RanBP10-HEp-2 cells infected with R7910 (ΔICP0) or R7911 (ΔICP0-repair) at an MOI of 5 for 18 h. In these ChIP assays, we used an antibody to histone H3, since H3 has been reported to be a marker for total histone levels (20, 50). The promoters of the viral ICP27, ICP8, and VP16 genes were assayed in the H3-immunoprecipitated chromatin using quantitative real-time PCR to measure the association of H3 with these promoters in infected cells. As shown in Fig. 13, the amount of DNA associated with histone H3 for all three viral gene promoters in sh-RanBP10/RanBP10-HEp-2cells infected with R7911 (ΔICP0-repair) was less than that in shRanBP10-HEp-2 cells infected with R7911 (ΔICP0-repair) and in sh-RanBP10/RanBP10-HEp2 cells infected with R7910 (ΔICP0). RanBP10 knockdown increased the amount of histone H3 associated with the three viral gene promoters in R7910 (ΔICP0)-infected cells more than in R7911 (ΔICP0-repair)-infected cells, and the ICP0-null mutation increased the association of histone H3 with the three viral gene promoters in sh-RanBP10-HEp-2 cells more than in sh-RanBP10/RanBP10-HEp-2 cells (Fig. 13). These results were in agreement with the effects of RanBP10 knockdown and the ICP0-null mutation on viral replication and on the accumulation of ICP27, ICP8, and VP16 mRNAs and proteins, as shown in Fig. 7 to 12. In these experiments, viral promoter DNAs immunoprecipitated with normal rabbit IgG were not detected (Fig. 13). These results indicated that RanBP10 was required for efficient reduction of histone association with the ICP27, ICP8, and VP16 gene promoters, and although both RanBP10 and ICP0 were required for chromatin remodeling at these HSV-1 promoters, each could partially compensate for the absence of the other.

FIG 13.

Effect of RanBP10 and/or ICP0 on histone H3 association with viral promoters. sh-RanBP10-HEp-2 and sh-RanBP10/RanBP10-HEp-2 cells were infected with R7910 (ΔICP0) or R7911 (ΔICP0-repair) at an MOI of 5 for 18 h. Chromatin immunoprecipitation (ChIP) was carried out with anti-histone H3 antibody or normal rabbit IgG. Immunoprecipitated DNA was quantitated by real-time PCR and the amounts of ICP27, ICP8, and VP16 promoters in the immunoprecipitated DNAs were determined. Immunoprecipitated DNAs with normal rabbit IgG were not detected in this study. Data are representative of three independent experiments.

DISCUSSION

Tandem affinity purification of transiently expressed HSV-1 ICP0 in HEK293T cells coupled with mass spectrometry-based proteomics technology identified a putative interaction between ICP0 and cell protein RanBP10. The interaction of ICP0 with RanBP10 was confirmed by reciprocal coimmunoprecipitation studies with cells transiently overexpressing MEF-tagged ICP0 and/or HA-tagged RanBP10. The interaction of ICP0 with RanBP10 was also confirmed by studies in HSV-1-infected cells: (i) HA-tagged RanBP10 coprecipitated with ICP0 in lysates of wild-type HSV-1(F)-infected cells stably expressing HA-tagged RanBP10, (ii) MEF-tagged ICP0 coprecipitated with HA-tagged RanBP10 in lysates of YK322 (MEF-ICP0)-infected cells stably expressing HA-tagged RanBP10, and (iii) ICP0 colocalized with HA-tagged RanBP10 in discrete nuclear domains in wild-type HSV-1(F)-infected cells stably expressing HA-tagged RanBP10. Thus, we have identified RanBP10 as a novel ICP0-interacting cell protein in this study. Our results also indicated that ICP0 interacted with RanBP10 in the absence of other HSV-1 proteins.

In this study, we also showed that RanBP10 was required for efficient HSV-1 replication, based on the results that RanBP10 knockdown reduced the replication of wild-type HSV-1, the ICP0-null mutant virus, and the UL41-enzyme-dead mutant virus. Thus, we also identified RanBP10 as a novel cell protein that promoted HSV-1 replication. We noted that the effect of RanBP10 knockdown on replication of the ICP0-null mutant virus was much greater than on replication of wild-type HSV-1 and the UL41-enzyme-dead mutant virus. A reciprocal effect was observed: the ICP0-null mutation reduced HSV-1 replication in RanBP10 knockdown cells much more than it did in control cells. In agreement with the effect of RanBP10 knockdown on replication of wild-type HSV-1 and the ICP0-null mutant virus, RanBP10 knockdown and the ICP0-null mutation both reduced the accumulation of IE gene ICP27, E gene ICP8, and L gene VP16 mRNAs and proteins, and these effects of RanBP10 knockdown and the ICP0-null mutation were synergistic. Similarly, RanBP10 knockdown and the ICP0-null mutation both increased histone H3 association with the ICP27, ICP8, and VP16 gene promoters, and these effects of RanBP10 knockdown and the ICP0-null mutation also appeared to be synergistic. Based on all of these results, we hypothesized that ICP0 and RanBP10 functioned redundantly in HSV-1-infected cells and that ICP0 and RanBP10 synergistically promoted viral gene expression by regulating viral chromatin remodeling for efficient viral replication. In support of this hypothesis are the following: (i) RanBP10 has been reported to be a transcriptional coactivator (30), and (ii) we showed that RanBP10 was recruited to HSV-1 replication compartments where viral DNA replication and transcription take place in HSV-1-infected cells, based on the observation that RanBP10 colocalized with ICP8, a marker of HSV-1 replication compartments, in HSV-1-infected cells. However, we cannot eliminate the possibility that a currently unidentified synergistic function(s) of ICP0 and RanBP10, other than in viral chromatin remodeling and in viral gene expression, is involved in HSV-1 gene expression and replication, respectively.

The mechanism(s) by which ICP0 and RanBP10 function redundantly and synergistically in viral chromatin remodeling, gene expression, and replication remains to be elucidated. A possible explanation for ICP0 and RanBP10 synergy and redundancy may be if they both function in a macromolecular protein complex to regulate viral chromatin remodeling and promote viral gene expression and replication in HSV-1-infected cells, as reported for cellular DNA methyltransferases DNMT1 and DNMT3b (51). Both DNMT1 and DNMT3b are components of epigenetic repressor complexes and redundantly and cooperatively regulate chromatin remodeling in human cancer cells (52, 53). In support of this hypothesis, ICP0 has been reported to interact with various cellular and HSV-1 proteins (3, 4, 22). In addition, RanBP10 may act as a coactivator of both ICP0 and another not yet identified viral and/or cellular regulatory protein(s) that may be a component of the epigenetic protein complex containing RanBP10 and ICP0. In agreement with this possibility, it has been reported that ICP0 interacts with several HSV-1 proteins, some of which are known to regulate HSV-1 gene expression together with ICP0 (54–58).

In conclusion, we identified RanBP10 as a novel ICP0-binding cell protein and showed that ICP0 and RanBP10 redundantly and synergistically regulated viral chromatin remodeling, gene expression, and replication. Thus far, in addition to RanBP10, ICP0 has been suggested to regulate viral chromatin remodeling by interacting with and/or modulating expression of host cell chromatin modifying factors, including CoREST/REST/HDAC/LSD1, CLOCK/BMAL1, and hDaxx/ATRX (4, 10, 21–23). Further studies to determine the contribution of each of the cellular factors or complexes in ICP0-mediated viral chromatin modification and replication will be of interest and are under way in this laboratory.

Funding Statement

MEXT and the Japan Agency for Medical Research and Development (AMED), contract research fund for the Program of Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), provided funding to Yasushi Kawaguchi. The Takeda Science Foundation provided grants to Akihisa Kato, Jun Arii, and Yasushi Kawaguchi. The Mitsubishi Foundation provided a grant to Yasushi Kawaguchi.