The targeted cytosolic degradation of class I histone deacetylases is essential for efficient alphaherpesvirus replication

24 Viral infection triggers a robust DNA damage response (DDR), reshaping the host 25 chromatin landscape to facilitate viral replication. Here, we uncover a novel 26 mechanism by which alphaherpesviruses exploit the DDR pathway. We demonstrate d 27 that herpes simplex virus 1 (HSV -1) and pseudorabies virus (PRV) induce d selective 28 degradation of class I histone deacetylases (HDAC1/2), leading to histone 29 hyperacetylation and subsequent DDR activation. Strikingly, viral infection promote d 30 nuclear expor t of HDAC1/2, followed by MDM2 -mediated K63 -linked 31 polyubiquitination and proteasomal degradation in the cytoplasm. Pharmacological 32 inhibition of either DDR signaling or HDAC1/2 nuclear export significantly affected 33 viral replication in vitro and in vivo . Our findings reveal a unique viral strategy to 34 hijack host epigenetic regulation for efficient replication and identify potential 35 therapeutic targets for alphaherpesvirus infections. 36

Alphaherpesviruses; HDACs; MDM2; Ubiquitination; DNA damage 37 response. 38 39 40 41 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint

42 HSV-1 is a widespread neurotropic virus that infects more than two -thirds of the 43 global population under the age of 50 [1, 2] . After initial infection of mucosal 44 epithelia and neuronal tissues, HSV -1 establishes lifelong latency within sensory 45 ganglia [1, 3]. Clinically, HSV-1 infection can present as oral or genital lesions and, in 46 severe instances, may progress to herpes simplex encephalitis —a potentially fatal 47 inflammation of the brain [4, 5]. Emerging evidence suggests a plausible association 48 between HSV -1 and neurodegenerative conditions such as Alzheimer’s disease, 49 linking chronic neuroinflamma tion and neuronal impairment to persistent viral 50 infection [6, 7]. To sustain lifelong infection, HSV-1 employs sophisticated strategies 51 to evade host immune defenses and maintain latent reservoirs. 52 Epigenetic regulation plays a critical role in governing HSV-1 latency and reactivation 53 [8, 9]. During latency, the virus coopts host histone deacetylases (HDACs) to enforce 54 viral genomic silencing through chromatin condensation [10]. Although HDAC1 and 55 HDAC2 are central to chromatin stability and DNA damage response (DDR), their 56 roles during alphaherpesvirus infection appear complex and context-dependent. While 57 these enzymes contribute to viral repression during latency [11], their potential 58 involvement in promoting lytic replication —whether through degradation or 59 functional inactivation—has remained inadequately explored. 60 Histone acetylation, a pivotal epigenetic modification that modulates chromatin 61 accessibility and transcriptional activity, is dynamically regulated by histone 62 acetyltransferases (HATs), which add acetyl groups to activate gene expression, and 63 HDACs, which remove these groups to enforce repression [12, 13] . The HDAC 64 family is categorized into four classes: Class I (HDAC1, 2, 3, 8), Class IIa/b, Class III 65 (sirtuins), and Class IV (HDAC11) [14]. Among these, Class I HDACs —particularly 66 HDAC1 and HDAC2—are indispensable for chromatin remodeling, genomic integrity, 67 and immune modulation [15-17]. These enzymes contribute to host antiviral defense 68 by fine-tuning the expression of immune-related genes. Nevertheless, the mechanisms 69 through which HSV-1 overcomes this transcriptional repression remain incompletely 70 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint understood. 71 HDAC1 serves as a key regulator of chromatin dynamics and innate immune 72 signaling, interfacing with critical pathways such as NF -κB, JAK-STAT, and Toll-like 73 receptor cascades to shape antiviral responses [18-20]. For example, in lung epithelial 74 cells, HDAC1 facilitates STAT1 phosphorylation and enhances interferon -stimulated 75 gene (ISG) activation, thereby restricting influenza A virus replication [21, 22]. This 76 underscores the dual function of HDAC1 in both epigenetic control and immune 77 regulation. However, the specific strategies used by HSV -1 to manipulate 78 HDAC1/2—particularly through ubiquitin-mediated degradation—to circumvent host 79 immunity and enhance viral replication have not been clearly defined. 80 This study reveals that HSV -1 induces the degradation of HDAC1/2 via an 81 MDM2-dependent ubiquitination mechanism, resulting in elevated histone acetylation, 82 chromatin relaxation, and activation of DNA damage response pathways that 83 collectively enhance viral replication. These findings offer novel insights into the 84 epigenetic subversion strategies employed by HSV -1 and underscore the therapeutic 85 potential of targeting the HDAC –MDM2 axis in the treatment of herpesvirus 86 infections. 87 88

89 Mice 90 Female C57BL/6J mice (6 –8 weeks old) were purchased from the Experimental 91 Animal Center of Zhengzhou University (Zhengzhou, China) and housed in 92 specific-pathogen-free facilities under controlled environmental conditions, including 93 a 12-hour light-dark cycle and a temperature of 22°C. Following 15 days of infection, 94 euthanasia was performed on the animals by administering pentobarbital so dium 95 intravenously (90 mg/kg) in compliance with the recommendations of the Chinese 96 Association for Laboratory Animal Sciences. The liver was excised for 97 immunoblotting analysis, and the remaining bodies were disposed of harmlessly. All 98 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint procedures were in accordance. 99 Reagents and plasmids 100 Reagents were sourced as follows: Leptomycin B (HY -16909), MG-132 (HY-13259), 101 3-MA (HY-19312), cycloheximide (HY-12320), and chloroquine (HY -17589A) were 102 purchased from MedChemExpress; berzosertib (S7102) was purchased from Selleck; 103 DMSO (W387520) was purchased from Sigma -Aldrich. Antibodies against CHK1 104 (25887-1-AP), CHK2 (13954 -1-AP), RAD51 (14961 -1-AP), β -actin (66009 -1-lg), 105 P53 (10442 -1-AP), HDAC1 (10197 -1-AP), HDAC2 (12922 -3-AP), HDAC4 106 (17449-1-AP), HDAC6 (12834 -1-AP), HDAC11 (67949 -1-Ig), and EGFP 107 (50430-2-AP) were purchased from Proteintech; antibodies against p -P53 (9286), 108 p-ATM (13050), ATM (2873), ATR (13934), p -ATR (2853), p -CHK1 (12302), 109 p-CHK2 (2197), γ-H2AX (80312), H3 (4499), H4K8ac (2594), H4K12ac (13944), H4 110 (13919), H2AX (7631), H3K9ac (4658), H3K27ac (8173), UB (20326), and MDM2 111 (86934) were purchased from Cell Signaling Technology; H3K56ac antibody (07-677) 112 was purchased from Millipore; ICP4 antibody (ab6514) was purchased from Abcam; 113 ICP0 antibody (sc -53070) was purchased from Santa Cruz Biotechnology; FLAG 114 antibody (F7425) was purchased from Sigma -Aldrich; HA antibody (A00169) was 115 purchased from GenScript. 116 HDAC1 and HDAC2 coding sequences were amplified from HEK293 cell cDNA and 117 cloned into p3×FLAG -CMV-10 to generate FLAG -tagged expression constructs. 118 MDM2 coding sequence was amplified and inserted into pEGFP -C1 to produce 119 EGFP-MDM2. Site -directed mutagenesis was performed using the QuikChange 120 Site-Directed Mutagenesis Kit (Agilent Technologies, 200523) per the manufacturer’s 121 instructions to generate: FLAG-HDAC1 K8R, FLAG-HDAC1 K10R, FLAG-HDAC1 122 K31R, FLAG- HDAC1 K50R, FLAG -HDAC1 K58R, FLAG -HDAC1 K66R, 123 FLAG-HDAC1 K74R, FLAG-HDAC1 K89R, FLAG-HDAC2 K75R, FLAG-HDAC1 124 (aa 1 –329), FLAG -HDAC1 (aa 329 –482), FLAG -HDAC1 (aa 1 –100), 125 FLAG-HDAC1 (aa 100 –329), and EGFP -MDM2∆RING (aa 1 –435). HA -tagged 126 ubiquitin constructs [HA-UB (WT), HA-UB (K48), HA-UB (K63)] were provided by 127 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint Dr. Bo Zhong (College of Life Sciences, Wuhan University, China). 128 Cells 129 Cell lines (HeLa/ATCC C L-82, Vero/ATCC CL -81, 3D4/21/ATCC CRL -2843, 130 HEK293T/ATCC CRL-11268) were cultured in DMEM or RPMI 1640 supplemented 131 with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L -glutamine 132 at 37°C under 5% CO₂ in a humidified incubator. For trans ient transfections, DNA 133 constructs were delivered to HeLa and HEK293T cells using Lipofectamine 3000 134 (Invitrogen, L3000001) per the manufacturer’s protocol. 135 To generate shRNA- mediated knockdown cell lines, HEK293T cells were 136 co-transfected with control or gene-specific shRNA (Table S1), packaging plasmid 137 psPAX2, and envelope plasmid pMD2.G. Post -transfection (6 h), medium was 138 replaced, and lentiviral particles were harvested at 48 hours. Parental cells were 139 infected with viral supernatant and selected with puromycin to establish stable 140 knockdown lines 141 Viruses 142 HSV-1 strain F (provided by Dr. Chun -Fu Zheng, University of Calgary, Canada) and 143 PRV-QXX were propagated per established protocols [23]. Viral titers were 144 determined by plaque assays in Vero cells. For in vitro infections, cells were infected 145 with HSV-1 or PRV-QXX at an MOI of 1. For in vivo studies, mice were intranasally 146 inoculated with HSV-1 (1 × 10⁶ pfu/mouse). 147 Immunoblotting, ubiquitination and Co-immunoprecipitation (Co-IP) 148 For immunoblotting, cells were lysed in RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 149 mM NaCl, 1% Triton X -100, 1% sodium deoxycholate, 0.1% SDS, 2 mM MgCl₂] 150 supplemented with protease/phosphatase inhibitors. Proteins were resolve d by 151 SDS-PAGE, transferred to PVDF membranes, and blocked with 5% nonfat milk in 152 TBST (1 hour, RT). Membranes were incubated with primary antibodies (overnight, 153 4°C), then HRP-conjugated secondary antibodies (1 hour, RT). Signals were detected 154 using Laminate Crescendo Western HRP Substrate (Millipore, Cat. No. WBLUR0500) 155 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint on a GE AI600 imager. 156 For ubiquitination assay, cells were lysed in IP buffer [50 mM Tris-HCl (pH 7.4), 150 157 mM NaCl, 1% NP -40, 1% sodium deoxycholate, 5 mM EDTA, 5 mM EGTA] and 158 clarified (16,000 × g, 10 minutes, 4°C). Subsequently, 900 μl aliquots were incubated 159 with 40 μl of a 1:1 slurry of Sepharose beads conjugated to anti -HDAC1 or 160 anti-FLAG mouse monoclonal antibodies (4°C, 4 hours). Beads were washed four 161 times with IP buffer, eluted in SDS sample buffer (10 minutes, boiling), and analyzed 162 by immunoblotting. 163 For Co-IP assay, cells were lysed in Co-IP buffer [50 mM Tris-HCl (pH 7.4), 150 mM 164 NaCl, 1% NP-40, 5 mM EDTA, 5 mM EGTA] and clarified (16,000 × g, 10 minutes, 165 4°C). Aliquots (900 μl ) were incubated with 40 μl of a 1:1 slurry of Sepharose beads 166 conjugated to IgG (GE Healthcare) or anti-FLAG mouse monoclonal antibody (4°C, 4 167 hours). Beads were washed three times with Co -IP buffer, eluted in SDS sample 168 buffer (10 minutes, boiling), and analyzed by immunoblotting. 169 Immunofluorescence assay 170 Cells were fixed in 4% paraformaldehyde at room temperature for 20 min on 171 coverslips in 12 -well plates. Following three washes with PBS, cells were 172 permeabilized with 0.2% Triton X -100 for 20 min and subsequently blocked with 10% 173 FBS. Specific primary antibodies, diluted in 10% FBS, were then applied to the cells 174 and incubated for 1 h at room temperature. After three PBS washes, cells were 175 exposed to the appropriate secondary antibodies, also diluted i n 10% FBS, for 1 h at 176 room temperature. Nuclei were stained with DAPI for 5 min at room temperature, 177 mounted using Prolong Diamond (Invitrogen), and visualized using a Zeiss LSM 800 178 confocal microscope. 179 Comet assay 180 Cells were seeded in 6 -well plates and tr eated accordingly. Frosted microscope slides 181 were coated with 0.5% normal melting point agarose. A mixture of 10 μL DMEM 182 containing around 10,000 cells and 75 μL of 0.7% low melting point agarose was 183 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint layered onto the pre -coated agarose. An additional 75 μL of 0.7% low melting point 184 agarose formed a third layer. The slides were lysed in a buffer containing 10 mM 185 Tris-HCl, pH 10.0, 2.5 M NaCl, 100 mM Na2EDTA, 1% Triton X -100, and 10% 186 DMSO for 2 hours at 4°C. Post -lysis, the slides were treated with an electro phoresis 187 solution (300 mM NaOH, 1 mM Na2EDTA, pH >13) for 40 minutes, electrophoresed 188 at 20 V ( ∼300 mA) for 25 minutes, and neutralized with 0.4 mM Tris -HCl (pH 7.5). 189 Subsequently, cells were stained with propidium iodide (PI, 5 μg/mL) and examined 190 using a Zeiss LSM 800 confocal microscope. DNA damage was assessed based on tail 191 moment using CometScore software. 192 qRT-PCR analysis 193 Total RNA was isolated using TRIzol reagent (TaKaRa) and reverse -transcribed into 194 cDNA with the PrimeScript RT reagent kit (TaKaRa) . qRT-PCR was performed in 195 triplicate using SYBR Premix Ex Taq (TaKaRa) according to the manufacturer's 196 protocol. Expression levels were normalized to β -actin as an internal reference. 197 Melting curve analysis confirmed amplification specificity by verifying 198 single-product formation in all reactions. Relative gene expression was calculated 199 using the 2˗ΔΔCt method. Primer sequences are provided in Table S1 200 Plaque assay 201 Vero cells (seeded in six-well plates) were grown to confluence, infected with serially 202 diluted viruses (10⁻¹ –10⁻⁷; 1 hour, 37°C), and washed with PBS to remove residual 203 inoculum. DMEM containing 1% methylcellulose (4 mL/well) was added, and cells 204 were inc ubated for 4 –5 days. Post -incubation, cells were fixed with 4% 205 paraformaldehyde (15 minutes), stained with 1% crystal violet (30 minutes), and 206 plaques were quantified. 207 Statistical analysis 208 Statistical analyses were performed using GraphPad Prism 8 software. Comparisons 209 between two groups were evaluated using a two -tailed Student’s t-test. Significance 210 was defined as P < 0.05. Data are presented as mean ± SD of three independent 211 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint experiments. Kaplan -Meier survival curves were generated and analyzed for mouse 212 survival assessment. 213 214

215 HSV-1 infection promotes HDAC1/2 degradation and histone hyperacetylation. 216 Post-translational modifications of histones play a critical role in regulating chromatin 217 remodeling and gene expression. HDACs, a highly conserved enzyme family [21], 218 catalyze the removal of acetyl groups from histones, thereby promoting chromatin 219 condensation and transcriptional repression. To investigate the effect of HSV -1 220 infection on HDAC expression and histone acetylation, we exam ined the protein 221 levels of key HDAC isoforms following viral infection. Immunoblotting analysis 222 revealed a marked decrease in HDAC1 and HDAC2 protein levels in cells infected 223 with either HSV -1 or PRV , whereas the expression of HDAC4, HDAC6, and 224 HDAC11 remained unchanged (Fig. 1A, B). In contrast, qRT -PCR results showed no 225 alterations in HDAC1 and HDAC2 mRNA levels (Fig. 1C, D), suggesting that their 226 depletion is regulated at the post-translational level. 227 To assess the functional impact of HDAC1/2 reduction, we examined global histone 228 acetylation patterns. Site -specific immunoblotting demonstrated elevated acetylation 229 at multiple lysine residues, including H3K9, H3K27, H3K56, H4K8, and H4K12, in 230 HSV-1-infected HeLa cells (Fig. 1E). A similar hyperacetylation profile was observed 231 in PRV-infected 3D4/21 cells and in murine liver tissues infected with HSV-1 (Fig. 1F, 232 G). Together, these findings indicate that HSV-1 selectively degrades class I HDACs, 233 resulting in widespread histone hyperacetylation that fosters a chromatin state 234 conducive to viral replication. 235 HSV-1 infection induces DDR through HDAC1/2 depletion and histone 236 hyperacetylation. 237 Given that excessive histone acetylation can destabilize chromatin structure, we 238 sought to elucidate the mechanism by which this epigenetic modification activates the 239 DDR pathway. Accumulating evidence underscores the pivotal role of histone 240 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint modifications in maintaining genomic stability, with dynamic changes in chromatin 241 architecture being intimately associated with DDR initiation. To determine whether 242 HSV-1 infection induces DDR activation, we evaluated the phosphorylation of 243 γ-H2AX, a well -established ma rker of DNA double -strand breaks. 244 Immunofluorescence microscopy revealed a significant increase in γ -H2AX foci in 245 cells infected with HSV -1 or PRV (Fig. 2A, B). Comet assays further corroborated 246 enhanced DNA fragmentation under these conditions (Fig. 2C –F). Given the central 247 role of ATM/ATR signaling in the DDR, we proceeded to examine the activation of 248 key checkpoint kinases. Immunoblotting analysis confirmed phosphorylation of ATM, 249 ATR, Chk1, Chk2, H2AX, and RAD51 in HSV -1-infected HeLa cells, 3D4/21 cell s, 250 and murine liver tissues (Fig. 2G, H). 251 Moreover, pharmacological inhibition of ATR using berzosertib markedly reduced 252 viral titers in plaque assays (Fig. 2I). In vivo , administration of berzosertib 253 significantly improved survival rates in HSV -1-infected mice (Fig. 2J). These results 254 demonstrate that HSV -1 infection activates the DDR, likely mediated through 255 HDAC1/2 degradation and consequent histone hyperacetylation. This virus -induced 256 DDR activation underscores the profound impact of HSV -1 on host chrom atin 257 dynamics and genomic integrity. 258 259 HSV-1 promotes the ubiquitin-proteasome degradation of HDAC1/2. 260 To elucidate the mechanism responsible for HDAC1/2 degradation, we focused on 261 their post -translational regulation. Since HDAC1/2 mRNA levels were unchanged 262 after infection, we hypothesized that the reduction in HDAC1/2 protein is mediated 263 through enhanced proteolytic degradation. To identify the relevant degradation 264 pathway, HSV-1-infected HeLa cells were treated with either the proteasome inhibitor 265 MG-132 or autophagy inhibitors (3 -MA and chloroquine). Immunoblot ting analysis 266 indicated that only MG -132 rescued HDAC1/2 protein levels (Fig. 3A), confirming 267 that degradation occurs primarily via the proteasomal pathway. 268 To examine whether HSV-1 infection induced ubiquitination of HDAC1, endogenous 269 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint immunoprecipitation assays were performed, which revealed increased ubiquitination 270 of HDAC1 following infection (Fig. 3B). Subsequent ubiquitination assays using 271 wild-type (WT), K48 -only, and K63 -only ubiquitin mutants demonstrated that 272 HDAC1 undergoes K63 -linked—but not K48 -linked—polyubiquitination (Fig. 3C). 273 Using a series of HDAC1 truncation mutants, we mapped the ubiquitination site to the 274 N-terminal region (amino acids 1–100) (Fig. 3D–F). Site-directed mutagenesis further 275 identified lysine 74 (K74) as the critical residue required for ubiquitination (Fig. 3G –276 I). Sequence alignment showed that HDAC2 K75 corresponds to HDAC1 K74 (Fig. 277 3J), and a K75R mutation in HDAC2 similarly abolished ubiquitination (Fig. 3J). 278 Together, these results indicate that HSV -1 infection promotes K63 -linked 279 polyubiquitination of HDAC1/2 at conserved lysine residues, ultimately leading to 280 their proteasomal degradation. 281 282 MDM2 functions as the E3 ligase mediating HDAC1/2 ubiquitination and 283 degradation. 284 Having established the occurrence of K63 -linked ubiquitination (Fig. 3), we next 285 sought to identify the E3 ubiquitin ligase responsible. An RNAi screen targeting 286 candidate E3 ligases revealed MDM2 as the principal mediator of HDAC1/2 287 degradation. Knockdown of MDM2 —but not of PIRH2, KCTD11, CHFR, UHRF1, 288 or TRIM46 —effectively prevented HDAC1/2 depletion (Fig. 4A, B). Co -IP assay 289 further confirmed an enhanced interaction between HDAC1 and MDM2 following 290 HSV-1 infection (Fig. 4C). 291 To characterize this interaction, we focused on the RING finger domain of MDM2, 292 which is essential for its E3 ligase activity. Co -IP analysis demonstrated that HSV -1 293 infection promoted binding between FLAG-HDAC1 and EGFP-MDM2, but not with 294 a RING -deleted mutant (E GFP-MDM2∆RING) (Fig. 4D), indicating that the 295 interaction was RING domain -dependent. To identify the region within HDAC1 296 required for binding, HeLa cells were co -transfected with EGFP -MDM2 and either 297 FLAG-HDAC1 or the ubiquitination -resistant mutant FLAG -HDAC1 K74R. 298 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint Infection with HSV-1 failed to induce binding between EGFP -MDM2 and the K74R 299 mutant (Fig. 4E), underscoring the essential role of lysine 74 in mediating 300 MDM2-dependent ubiquitination. 301 Ubiquitination assay further revealed that overexpression of EGFP-MDM2∆RING did 302 not enhance HSV -1-induced ubiquitination or degradation of HDAC1 (Fig. 4F). 303 Similarly, the FLAG-HDAC1 K74R mutant remained resistant to ubiquitination under 304 all experimental conditions (Fig. 4F). Collectively, these results demonstrate t hat 305 HSV-1 induces HDAC1/2 degradation through MDM2 -mediated K63 -linked 306 ubiquitination. 307 308 HSV-1 triggers cytoplasmic translocation of HDAC1 to facilitate its degradation. 309 To elucidate the mechanisms by which HSV -1 induces HDAC degradation, we 310 systematically analyzed the subcellular localization of HDAC1. In mock -infected 311 HeLa cells, HDAC1 was predominantly nuclear; however, HSV -1 infection triggered 312 a pronounced translocation of HDAC1 to the cytoplasm (Fig. 5A). This redistribution 313 was completely abolished by treatment with leptomycin B (LMB), a specific inhibitor 314 of CRM1-mediated nuclear export (Fig. 5A). 315 To determine whether translocation is functionally linked to degradation, we 316 conducted cycloheximide (CHX) chase assays. Degradation of both HDAC1 and 317 HDAC2 began approximately 12 hours after CHX treatment and was markedly 318 accelerated by HSV -1 infection (Fig. 5B). Importantly, LMB treatment restored 319 degradation kinetics to levels comparable to those in mock -infected cells (Fig. 5B). 320 Moreover, under LMB treatm ent, HSV -1 was unable to induce ubiquitination of 321 endogenous HDAC1 (Fig. 5C). 322 Immunoblotting analysis further indicated that LMB suppressed HSV -1–induced 323 phosphorylation of p53 and H2AX (Fig. 5E), confirming that CRM1 -dependent 324 nuclear export is essential for MDM2-mediated HDAC1 degradation. Notably, LMB 325 treatment—which inhibits CRM1 -dependent export and consequently prevents 326 HDAC1 degradation —significantly impaired viral replication (Fig. 5F). Together, 327 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint these findings demonstrate that HSV -1 infection promo tes cytoplasmic translocation 328 of HDAC1, facilitating its ubiquitination and subsequent proteasomal degradation. 329 330

331 HSV-1 manipulates host cellular pathways to facilitate its replication, latency, and 332 reactivation [2]. In this study, we demonstrate that alphaherpesvirus targets class I 333 histone deacetylases HDAC1 and HDAC2 for MDM2 -mediated K63 -linked 334 polyubiquitination and subsequent proteasomal degradation. This degradation induces 335 histone hyperacetylation, chromatin relaxation, and enhanced viral 336 replication—revealing a mechanism through which alphaherpesvirus reprograms the 337 host epigenetic landscape to optimize viral replication (Fig. 6). 338 Unlike human cytomegalovirus, which upregulates HDAC expression to suppress 339 host immunity [24], HSV-1 promotes the degradation of HDAC1/2 to establish a 340 chromatin environment conducive to viral transcription. This highlights a fundamental 341 divergence in epigenetic strategies among herpesviruses. Our findings indicate that 342 virus-directed degradation of HDAC1/2 occurs specifically during lytic replication 343 [25], suggesting a dual role for these deacetylases: their presence supports latency, 344 whereas their degradation facilitates lytic replication. 345 We identified MDM2 [26] as the principal E3 ubiquitin ligase responsible for 346 ubiquitinating HDAC1 at lysine 74 and HDAC2 at lysine 75, leading to their 347 proteasomal degradation. This mechanism is distinct from other E3 ligases, including 348 PIRH2 [27] and TRIM46 [28]), and critically depends on the RING domain of MDM2. 349 While K63-linked ubiquitination is typically associated with non-proteolytic functions, 350 our work establishes its novel role in mediating HDAC1/2 degradation—uncovering a 351 previously unrecognized viral pathogenesis pathway. Targeting this axis using MDM2 352 inhibitors or inhibiting nuclear export of HDAC1/2 significantly restricts viral 353 replication, suggesting that host -directed antiviral strategies may help overcome 354 resistance to direct-acting agents. 355 Depletion of HDAC1/2 increases acet ylation at histone residues associated with open 356 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint chromatin and active transcription. Similar chromatin remodeling has been observed 357 in other viral infections [29], underscoring its conserved role in viral gene regulation. 358 Our results are consistent across multiple cell types, including HeLa, 3D4/21, and 359 murine liver, emphasizing the central importance of HDAC1/2 suppression in HSV -1 360 biology. Notably, HDAC1/2 degradation activates DDR, as indicated by increased 361 γ-H2AX foci and phosphorylation of ATM, ATR, Chk1, and Chk2. This aligns with 362 previous reports that HSV -1 exploits D DR signaling to enhance replication [30-32]. 363 Importantly, HDAC1/2 undergo CRM1 -dependent nuclear ex port prior to 364 degradation—a process inhibited by leptomycin B —highlighting the essential role of 365 subcellular localization in viral manipulation of host proteins. 366 HSV-1–mediated HDAC1/2 degradation likely supports viral replication through 367 multiple mechanisms, including evasion of nuclear antiviral sensors and stimulation 368 of viral gene expression. Targeting the MDM2–HDAC1/2 axis represents a promising 369 antiviral strategy. MDM2 inhibitors reduce HSV -1 titers [33] , suggesting that 370 modulating ubiquitin ation pathways could complement existing therapies. Beyond 371 herpesviruses, the MDM2–HDAC axis may be exploited by other DNA viruses (e.g., 372 HBV , HPV) that manipulate chromatin and DDR, indicating a possible common host 373 vulnerability. However, broad HDAC inhi bition may unintentionally enhance viral 374 replication, underscoring the need for targeted therapeutic approaches. 375 This study primarily employed in vitro models (e.g., HeLa and 3D4/21 cells), which 376 lack the neuronal environment essential for studying HSV -1 l atency. Future work 377 should validate these findings in primary neurons and trigeminal ganglia [34]. 378 Additional studies are also needed to determine whether HDAC1/2 degradation 379 occurs during reactivation or is limited to lytic infection [8], and to explore the 380 interplay between HDAC degradation, viral tegument proteins, and host stress 381 responses. In vivo evaluation of inhibitors targeting the MDM2/HDAC1 –2 pathway 382 will be crucial for assessing their therapeutic potential. 383 384 Author contributions 385 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint Sheng-Li Ming and Meng-Hua Du : Data curation, Formal analysis, Investigation, 386 Methodology, Validation, Visualization, Writing – original draft; Jia -Ming Yang: Data 387 curation, Formal analysis, Investigation, Validation; Ya -Di Guo: Data curation, Formal 388 analysis, Investigation, Validation, Visualization; Jia-Jia Pan: Data curation, Formal analysis, 389 Investigation, Methodology, Visualization; Wei -Fei Lu: Data curation, Formal analysis, 390 Investigation; Jiang Wang: Methodology, Visualization, Project administration, Supervision, 391 Writing – review & editing; Lei Zeng and Bei -Bei Chu: Conceptualization, Data curation, 392 Funding acquisi -tion, Investigation, Methodology, Project administration, Resources, 393 Supervision, Validation, Writing – original draft, Writing – review & editing. 394 395 Declaration of competing interest 396 The authors declare that they have no competing interests. 397 398 Ethics statement 399 All procedures involving animals in this study received ethical approval from the 400 Institutional Animal Care and Use Committee (IACUC) of Henan Agricultural 401 University (Permit Number: HNND20 20031008). This research was performed in 402 accordance with the regulations specified in the Guide for the Care and Use of 403 Laboratory Animals established by the Ministry of Science and Technology of China 404 405 Funding 406 This research was supported by grants from the National Key R&D Program of China 407 (2021YFD1301200 and 2023YFD1801600), the China Postdoctoral Science 408 Foundation (2025M770267, GZC20240430). 409 410 Data Availability Statement 411 The datas that support the findings of this study are openlyavailable in “Mendeley Data , V3” 412 at https://data.mendeley.com/datasets/yg5fgtvxzk/3, doi: 10.17632/yg5fgtvxzk.3, reference 413 number[35]. 414 415 Declaration of generative AI and AI-assisted technologies in the writing process 416 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint This study affirms that neither generative artificial intelligence nor any other 417 AI-assisted technologies were employed in the preparation of this manuscript. 418 419

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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint Figure legends 518 Figure 1. Viral infection induces degradation of class I HDACs and 519 hyperacetylation of histones H3 and H4. 520 (A) Immunoblotting analysis of the indicated HDACs in HeLa cells infected with 521 HSV-1 (MOI = 1) for the indicated time points. 522 (B) Immunoblotting analysis of the indicated HDACs in 3D4/21 cells infected with 523 PRV-QXX (MOI = 1) for the indicated time points. 524 (C and D) qRT-PCR analysis of HDAC1 and HDAC2 mRNA levels in HSV-1-infected 525 HeLa cells (C) or PRV -QXX-infected 3D4/21 cells (D) (MOI = 1). Data are mean ± 526 SEM; n = 3. **P < 0.01, ***P < 0.001. 527 (E) Immunoblotting of the indicated proteins in HeLa cells from infected with HSV-1 528 (MOI = 1) for the indicated time points. 529 (F) Immunoblotting of the indicated proteins in 3D4/21 cells from infected with 530 PRV-QXX (MOI = 1) for the indicated time points. 531 (G) Immunoblotting analysis of the indicated proteins in liver tissues from mice 532 mock-infected or infected with HSV -1 (1 × 10⁶ pfu per mouse) at 5 days 533 post-infection (n = 3). 534 535 Figure 2. Viral infection triggers chromatin dysfunction and DDR activation via 536 HDAC degradation. 537 (A and B ) Immunofluorescence staining of γ -H2AX (red) and H2AX ( green) in 538 HSV-1-infected HeLa cells (A) or PRV -infected 3D4/21 cells (B) (MOI = 1). Scale 539 bars: 10 μm. 540 (C and D ) Comet assay assessing DNA damage in HeLa cells infected with HSV -1 541 (MOI = 1). Data are mean ± SEM; n = 40 cells/group. **P < 0.01, ***P < 0.001. 542 (E and F ) Comet assay assessing DNA damage in 3D4/21 cells infected with PRV 543 (MOI = 1) at the indicated time points. Data are mean ± SEM; n = 40 cells/group. **P 544 < 0.01, ***P < 0.001. 545 (G and H) Immunoblotting analysis of DDR markers (p -ATM, p -ATR, p -Chk1, 546 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint p-Chk2, γ-H2AX) and viral ICP4/EP0 in HSV -1-infected HeLa cells (G) or 547 PRV-QXX-infected 3D4/21 cells (H) (MOI = 1). 548 (I) Viral titers in HSV -1-infected HeLa cells (MOI = 1) treated with berzosertib (50 549 nM) or vehicle. Data are mean ± SEM; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 550 (J) Survival curves of HSV -1-infected mice (1 × 10⁶ PFU/mouse) treated with 551 berzosertib (20 mg/kg) or vehicle (n = 12/group), ***P < 0.001. 552 553 Figure 3. Viral infection induces K63-linked ubiquitination of HDAC1/2. 554 (A) Immunoblotting analysis of the indicated proteins in HeLa cells either 555 mock-infected or infected with HSV -1 (MOI = 1), treated with vehicle, 3 -MA (10 556 μM), MG-132 (10 μM), or chloroquine (10 μM) for the indicated times. 557 (B) Ubiquitination assay of HDAC1 in HeLa cells eith er mock -infected or infected 558 with HSV-1 (MOI = 1) at 24 hpi. 559 (C) Ubiquitination assay of FLAG -HDAC1 in HeLa cells transfected with 560 HA-ubiquitin variants (WT, K48, K63), and either mock -infected or infected with 561 HSV-1 (MOI = 1) at 24 hpi. 562 (D) Schematic representation of HDAC1 deletion mutants. 563 (E-I) Ubiquitination assays of FLAG -HDAC1 mutant variants in HeLa cells either 564 mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. 565 (J) Ubiquitination assays of FLAG -HDAC2 variants (WT and K75R) in HeLa cells 566 either mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. 567 568 Figure 4. MDM2 mediates viral -induced HDAC1/2 degradation via K74/K75 569 ubiquitination. 570 (A) qRT-PCR analysis validation of E3 ligase knockdown efficiency (shPIRH2, 571 shKCTD11, shMDM2, shCHFR, shUHRF1, shTRIM46) in HeLa cells. Data are mean 572 ± SEM; n = 3. ***P < 0.001. 573 (B) Immunoblotting analysis of HDAC1/2 stability in E3 ligase -knockdown HeLa 574 cells infected with HSV-1 (MOI = 1). 575 (C) Co-IP assay of endogenous HDAC1 -MDM2 interaction in HSV -1-infected HeLa 576 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint cells (MOI = 1; 24 hpi). 577 (D) Co-IP assay of FLAG -HDAC1 with EGFP -MDM2 (WT or ΔRING) in 578 HSV-1-infected HeLa cells (MOI = 1; 24 hpi). 579 (E) Co-IP assay of FLAG -HDAC1 (WT or K74R) with EGFP -MDM2 in 580 HSV-1-infected HeLa cells (MOI = 1; 24 hpi). 581 (F) Ubiquitination assays of FLAG -HDAC1 (WT or K74R) co -expressed with 582 EGFP-MDM2 (WT or ΔRING) in HSV-1-infected HeLa cells (MOI = 1; 24 hpi). 583 584 Figure 5. Cytoplasmic translocation enables MDM2 -mediated HDAC1 585 degradation. 586 (A) Immunofluorescence staining of HDAC1 ( red) and viral ICP4 ( green) in 587 HSV-1-infected HeLa cells (MOI = 1) ± leptomycin B (LMB; 10 nM; 24 h). Scale bar: 588 10 μm. 589 (B) CHX chase assay measuring HDAC1/2 degradation kinetics in HSV -1-infected 590 HeLa cells (MOI = 1) ± LMB (10 μM). 591 (C) Ubiquitination assay of HDAC1 in HSV-1-infected HeLa cells (MOI = 1) ± LMB 592 (10 μM; 24 h). 593 (D) Immunofluorescence analysis of MDM2 (red) and ICP4 (green) in 594 HSV-1-infected HeLa cells (MOI = 1) ± LMB (10 μM; 24 h). Scale bar: 10 μm. 595 (E) Immunoblotting analysis of DDR markers (γ -H2AX) and HDAC1/2 in 596 HSV-1-infected HeLa cells (MOI = 1) ± LMB (10 μM). 597 (F) Viral titers in HSV -1-infected HeLa cells (MOI = 1) ± LMB (10 μM). Data are 598 mean ± SEM; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 599 600 Figure 6. A schematic model showing the targeted cytosolic degradation of class I 601 histone deacetylases is essential for efficient alphaherpesvirus replication. 602 603 604 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint