{"paper_id":"2d4e01eb-dc99-4f2b-b45e-1b8de2a17208","body_text":"The targeted cytosolic degradation of class I histone deacetylases is essential for 1 \nefficient alphaherpesvirus replication  2 \nSheng-Li Ming 1,2,3#, Meng-Hua Du 1,2,3#, Jia-Ming Yang 6#, Ya -Di Guo 1,2,3, Jia-Jia 3 \nPan1,2,3, Wei-Fei Lu1,2,3, Jiang Wang1,2,3,5, Lei Zeng1,2,3*, Bei-Bei Chu1,2,3,4,5* 4 \n 5 \n1College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, 6 \nHenan Province, China 7 \n2Key Laboratory of Animal Biochemistry and Nu trition, Ministry of Agriculture and 8 \nRural Affairs, China 9 \n3Key Laboratory of Animal Growth and Development of Henan Province, Henan 10 \nAgricultural University, Zhengzhou 450046, Henan Province, China 11 \n4International Joint Research Center of National Animal Immunology, Henan 12 \nAgricultural University, Zhengzhou 450046, Henan Province, China 13 \n5Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, 14 \nZhengzhou 450046, Henan Province, China 15 \n6State Key Laboratory of Membrane Biology, School of Pharma ceutical Sciences, 16 \nTsinghua University, Beijing 100084, China 17 \n#These authors contributed equally  18 \n 19 \n*Correspondence: zenglei@henau.edu.cn (L. Zeng); chubeibei@henau.edu.cn (B.-B. 20 \nChu). 21 \n 22 \n  23 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nAbstract 24 \nViral infection triggers a robust DNA damage response (DDR), reshaping the host 25 \nchromatin landscape to facilitate viral replication. Here, we uncover a novel 26 \nmechanism by which alphaherpesviruses exploit the DDR pathway. We demonstrate d 27 \nthat herpes simplex virus 1 (HSV -1) and pseudorabies virus (PRV) induce d selective 28 \ndegradation of class I histone deacetylases (HDAC1/2), leading to histone 29 \nhyperacetylation and subsequent DDR activation. Strikingly, viral infection promote d 30 \nnuclear expor t of HDAC1/2, followed by MDM2 -mediated K63 -linked 31 \npolyubiquitination and proteasomal degradation in the cytoplasm. Pharmacological 32 \ninhibition of either DDR signaling or HDAC1/2 nuclear export significantly affected 33 \nviral replication in vitro and in vivo . Our findings reveal a unique viral strategy to 34 \nhijack host epigenetic regulation for efficient replication and identify potential 35 \ntherapeutic targets for alphaherpesvirus infections. 36 \nKeywords: Alphaherpesviruses; HDACs; MDM2; Ubiquitination; DNA damage 37 \nresponse. 38 \n 39 \n 40 \n  41 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nIntroduction 42 \nHSV-1 is a widespread neurotropic virus that infects more than two -thirds of the 43 \nglobal population under the age of 50  [1, 2] . After initial infection of mucosal 44 \nepithelia and neuronal tissues, HSV -1 establishes lifelong latency within sensory 45 \nganglia [1, 3]. Clinically, HSV-1 infection can present as oral or genital lesions and, in 46 \nsevere instances, may progress to herpes simplex encephalitis —a potentially fatal 47 \ninflammation of the brain  [4, 5]. Emerging evidence suggests a plausible association 48 \nbetween HSV -1 and neurodegenerative conditions such as Alzheimer’s disease, 49 \nlinking chronic neuroinflamma tion and neuronal impairment to persistent viral 50 \ninfection [6, 7]. To sustain lifelong infection, HSV-1 employs sophisticated strategies 51 \nto evade host immune defenses and maintain latent reservoirs. 52 \nEpigenetic regulation plays a critical role in governing HSV-1 latency and reactivation 53 \n[8, 9]. During latency, the virus coopts host histone deacetylases (HDACs) to enforce 54 \nviral genomic silencing through chromatin condensation  [10]. Although HDAC1 and 55 \nHDAC2 are central to chromatin stability and DNA damage response (DDR), their 56 \nroles during alphaherpesvirus infection appear complex and context-dependent. While 57 \nthese enzymes contribute to viral repression during latency  [11], their potential 58 \ninvolvement in promoting lytic replication —whether through degradation or 59 \nfunctional inactivation—has remained inadequately explored. 60 \nHistone acetylation, a pivotal epigenetic modification that modulates chromatin 61 \naccessibility and  transcriptional activity, is dynamically regulated by histone 62 \nacetyltransferases (HATs), which add acetyl groups to activate gene expression, and 63 \nHDACs, which remove these groups to enforce repression  [12, 13] . The HDAC 64 \nfamily is categorized into four classes: Class I (HDAC1, 2, 3, 8), Class IIa/b, Class III 65 \n(sirtuins), and Class IV (HDAC11)  [14]. Among these, Class I HDACs —particularly 66 \nHDAC1 and HDAC2—are indispensable for chromatin remodeling, genomic integrity, 67 \nand immune modulation  [15-17]. These enzymes contribute to host antiviral defense 68 \nby fine-tuning the expression of immune-related genes. Nevertheless, the mechanisms 69 \nthrough which HSV-1 overcomes this transcriptional repression remain incompletely 70 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nunderstood. 71 \nHDAC1 serves as a key regulator of chromatin dynamics and innate immune 72 \nsignaling, interfacing with critical pathways such as NF -κB, JAK-STAT, and Toll-like 73 \nreceptor cascades to shape antiviral responses [18-20]. For example, in lung epithelial 74 \ncells, HDAC1 facilitates STAT1 phosphorylation and enhances interferon -stimulated 75 \ngene (ISG) activation, thereby restricting influenza A virus replication  [21, 22]. This 76 \nunderscores the dual function of HDAC1 in both epigenetic control and immune 77 \nregulation. However, the specific strategies used by HSV -1 to manipulate 78 \nHDAC1/2—particularly through ubiquitin-mediated degradation—to circumvent host 79 \nimmunity and enhance viral replication have not been clearly defined. 80 \nThis study reveals that HSV -1 induces the degradation of HDAC1/2 via an 81 \nMDM2-dependent ubiquitination mechanism, resulting in elevated histone acetylation, 82 \nchromatin relaxation, and activation of DNA damage response pathways that 83 \ncollectively enhance viral replication. These findings offer novel insights into the 84 \nepigenetic subversion strategies employed by HSV -1 and underscore the therapeutic  85 \npotential of targeting the HDAC –MDM2 axis in the treatment of herpesvirus 86 \ninfections. 87 \n 88 \nMaterials and Methods  89 \nMice 90 \nFemale C57BL/6J mice (6 –8 weeks old) were purchased from the Experimental 91 \nAnimal Center of Zhengzhou University (Zhengzhou, China) and housed in 92 \nspecific-pathogen-free facilities under controlled environmental conditions, including 93 \na 12-hour light-dark cycle and a temperature of 22°C.  Following 15 days of infection, 94 \neuthanasia was performed on the animals by administering pentobarbital so dium 95 \nintravenously (90 mg/kg) in compliance with the recommendations of the Chinese 96 \nAssociation for Laboratory Animal Sciences. The liver was excised for 97 \nimmunoblotting analysis, and the remaining bodies were disposed of harmlessly. All 98 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nprocedures were in accordance. 99 \nReagents and plasmids 100 \nReagents were sourced as follows: Leptomycin B (HY -16909), MG-132 (HY-13259), 101 \n3-MA (HY-19312), cycloheximide (HY-12320), and chloroquine (HY -17589A) were 102 \npurchased from MedChemExpress; berzosertib (S7102) was purchased from Selleck; 103 \nDMSO (W387520) was purchased from Sigma -Aldrich. Antibodies against CHK1 104 \n(25887-1-AP), CHK2 (13954 -1-AP), RAD51 (14961 -1-AP), β -actin (66009 -1-lg), 105 \nP53 (10442 -1-AP), HDAC1 (10197 -1-AP), HDAC2 (12922 -3-AP), HDAC4 106 \n(17449-1-AP), HDAC6 (12834 -1-AP), HDAC11 (67949 -1-Ig), and EGFP 107 \n(50430-2-AP) were purchased from Proteintech; antibodies against p -P53 (9286), 108 \np-ATM (13050), ATM (2873), ATR (13934), p -ATR (2853), p -CHK1 (12302), 109 \np-CHK2 (2197), γ-H2AX (80312), H3 (4499), H4K8ac (2594), H4K12ac (13944), H4 110 \n(13919), H2AX (7631), H3K9ac (4658), H3K27ac (8173), UB (20326), and MDM2 111 \n(86934) were purchased from Cell Signaling Technology; H3K56ac antibody (07-677) 112 \nwas purchased from Millipore; ICP4 antibody (ab6514) was purchased from Abcam; 113 \nICP0 antibody (sc -53070) was purchased from Santa Cruz Biotechnology; FLAG 114 \nantibody (F7425) was purchased from Sigma -Aldrich; HA antibody (A00169) was 115 \npurchased from GenScript. 116 \nHDAC1 and HDAC2 coding sequences were amplified from HEK293 cell cDNA and 117 \ncloned into p3×FLAG -CMV-10 to generate FLAG -tagged expression constructs. 118 \nMDM2 coding sequence was amplified and inserted into pEGFP -C1 to produce 119 \nEGFP-MDM2. Site -directed mutagenesis was performed using the QuikChange 120 \nSite-Directed Mutagenesis Kit (Agilent Technologies, 200523) per the manufacturer’s 121 \ninstructions to generate: FLAG-HDAC1 K8R, FLAG-HDAC1 K10R, FLAG-HDAC1 122 \nK31R, FLAG- HDAC1 K50R, FLAG -HDAC1 K58R, FLAG -HDAC1 K66R, 123 \nFLAG-HDAC1 K74R, FLAG-HDAC1 K89R, FLAG-HDAC2 K75R, FLAG-HDAC1 124 \n(aa 1 –329), FLAG -HDAC1 (aa 329 –482), FLAG -HDAC1 (aa 1 –100), 125 \nFLAG-HDAC1 (aa 100 –329), and EGFP -MDM2∆RING (aa 1 –435). HA -tagged 126 \nubiquitin constructs [HA-UB (WT), HA-UB (K48), HA-UB (K63)] were provided by 127 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nDr. Bo Zhong (College of Life Sciences, Wuhan University, China). 128 \nCells  129 \nCell lines (HeLa/ATCC C L-82, Vero/ATCC CL -81, 3D4/21/ATCC CRL -2843, 130 \nHEK293T/ATCC CRL-11268) were cultured in DMEM or RPMI 1640 supplemented 131 \nwith 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L -glutamine 132 \nat 37°C under 5% CO₂ in a humidified incubator. For trans ient transfections, DNA 133 \nconstructs were delivered to HeLa and HEK293T cells using Lipofectamine 3000 134 \n(Invitrogen, L3000001) per the manufacturer’s protocol. 135 \nTo generate shRNA- mediated knockdown cell lines, HEK293T cells were 136 \nco-transfected with control or gene-specific shRNA (Table S1), packaging plasmid 137 \npsPAX2, and envelope plasmid pMD2.G. Post -transfection (6 h), medium was 138 \nreplaced, and lentiviral particles were harvested at 48 hours. Parental cells were 139 \ninfected with viral supernatant and selected with puromycin to establish stable 140 \nknockdown lines 141 \nViruses 142 \nHSV-1 strain F (provided by Dr. Chun -Fu Zheng, University of Calgary, Canada) and 143 \nPRV-QXX were propagated per established protocols  [23]. Viral titers were 144 \ndetermined by plaque assays in Vero cells. For in vitro infections, cells were infected 145 \nwith HSV-1 or PRV-QXX at an MOI of 1. For in vivo studies, mice were intranasally 146 \ninoculated with HSV-1 (1 × 10⁶ pfu/mouse). 147 \nImmunoblotting, ubiquitination and Co-immunoprecipitation (Co-IP) 148 \nFor immunoblotting, cells were lysed in RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 149 \nmM NaCl, 1% Triton X -100, 1% sodium deoxycholate, 0.1% SDS, 2 mM MgCl₂] 150 \nsupplemented with protease/phosphatase inhibitors. Proteins were resolve d by 151 \nSDS-PAGE, transferred to PVDF membranes, and blocked with 5% nonfat milk in 152 \nTBST (1 hour, RT). Membranes were incubated with primary antibodies (overnight, 153 \n4°C), then HRP-conjugated secondary antibodies (1 hour, RT). Signals were detected 154 \nusing Laminate Crescendo Western HRP Substrate (Millipore, Cat. No. WBLUR0500) 155 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\non a GE AI600 imager. 156 \nFor ubiquitination assay, cells were lysed in IP buffer [50 mM Tris-HCl (pH 7.4), 150 157 \nmM NaCl, 1% NP -40, 1% sodium deoxycholate, 5 mM EDTA, 5 mM EGTA] and 158 \nclarified (16,000 × g, 10 minutes, 4°C). Subsequently, 900 μl aliquots were incubated 159 \nwith 40 μl of a 1:1 slurry of Sepharose beads conjugated to anti -HDAC1 or 160 \nanti-FLAG mouse monoclonal antibodies (4°C, 4 hours). Beads were washed four 161 \ntimes with IP buffer, eluted in SDS sample buffer (10 minutes, boiling), and analyzed 162 \nby immunoblotting. 163 \nFor Co-IP assay, cells were lysed in Co-IP buffer [50 mM Tris-HCl (pH 7.4), 150 mM 164 \nNaCl, 1% NP-40, 5 mM EDTA, 5 mM EGTA] and clarified (16,000 × g, 10 minutes, 165 \n4°C). Aliquots (900 μl ) were incubated with 40 μl of a 1:1 slurry of Sepharose beads 166 \nconjugated to IgG (GE Healthcare) or anti-FLAG mouse monoclonal antibody (4°C, 4 167 \nhours). Beads were washed three times with Co -IP buffer, eluted in SDS sample 168 \nbuffer (10 minutes, boiling), and analyzed by immunoblotting. 169 \nImmunofluorescence assay 170 \nCells were fixed in 4% paraformaldehyde at room temperature for 20 min on 171 \ncoverslips in 12 -well plates. Following three washes with PBS, cells were 172 \npermeabilized with 0.2% Triton X -100 for 20 min and subsequently blocked with 10% 173 \nFBS. Specific primary antibodies, diluted in 10% FBS, were then applied to the cells 174 \nand incubated for 1 h at room temperature. After three PBS washes, cells were 175 \nexposed to the appropriate secondary antibodies, also diluted i n 10% FBS, for 1 h at 176 \nroom temperature. Nuclei were stained with DAPI for 5 min at room temperature, 177 \nmounted using Prolong Diamond (Invitrogen), and visualized using a Zeiss LSM 800 178 \nconfocal microscope. 179 \nComet assay 180 \nCells were seeded in 6 -well plates and tr eated accordingly. Frosted microscope slides 181 \nwere coated with 0.5% normal melting point agarose. A mixture of 10 μL DMEM 182 \ncontaining around 10,000 cells and 75 μL of 0.7% low melting point agarose was 183 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nlayered onto the pre -coated agarose. An additional 75 μL  of 0.7% low melting point 184 \nagarose formed a third layer. The slides were lysed in a buffer containing 10 mM 185 \nTris-HCl, pH 10.0, 2.5 M NaCl, 100 mM Na2EDTA, 1% Triton X -100, and 10% 186 \nDMSO for 2 hours at 4°C. Post -lysis, the slides were treated with an electro phoresis 187 \nsolution (300 mM NaOH, 1 mM Na2EDTA, pH >13) for 40 minutes, electrophoresed 188 \nat 20 V ( ∼300 mA) for 25 minutes, and neutralized with 0.4 mM Tris -HCl (pH 7.5). 189 \nSubsequently, cells were stained with propidium iodide (PI, 5 μg/mL) and examined 190 \nusing a Zeiss LSM 800 confocal microscope. DNA damage was assessed based on tail 191 \nmoment using CometScore software. 192 \nqRT-PCR analysis 193 \nTotal RNA was isolated using TRIzol reagent (TaKaRa) and reverse -transcribed into 194 \ncDNA with the PrimeScript RT reagent kit (TaKaRa) . qRT-PCR was performed in 195 \ntriplicate using SYBR Premix Ex Taq (TaKaRa) according to the manufacturer's 196 \nprotocol. Expression levels were normalized to β -actin as an internal reference. 197 \nMelting curve analysis confirmed amplification specificity by verifying  198 \nsingle-product formation in all reactions. Relative gene expression was calculated 199 \nusing the 2˗ΔΔCt method. Primer sequences are provided in Table S1 200 \nPlaque assay 201 \nVero cells (seeded in six-well plates) were grown to confluence, infected with serially 202 \ndiluted viruses (10⁻¹ –10⁻⁷; 1 hour, 37°C), and washed with PBS to remove residual 203 \ninoculum. DMEM containing 1% methylcellulose (4 mL/well) was added, and cells 204 \nwere inc ubated for 4 –5 days. Post -incubation, cells were fixed with 4% 205 \nparaformaldehyde (15 minutes), stained with 1% crystal violet (30 minutes), and 206 \nplaques were quantified. 207 \nStatistical analysis 208 \nStatistical analyses were performed using GraphPad Prism 8 software. Comparisons 209 \nbetween two groups were evaluated using a two -tailed Student’s t-test. Significance 210 \nwas defined as P < 0.05. Data are presented as mean ± SD of three independent 211 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nexperiments. Kaplan -Meier survival curves were generated and analyzed for mouse 212 \nsurvival assessment. 213 \n 214 \nResults 215 \nHSV-1 infection promotes HDAC1/2 degradation and histone hyperacetylation. 216 \nPost-translational modifications of histones play a critical role in regulating chromatin 217 \nremodeling and gene expression. HDACs, a highly conserved enzyme family  [21], 218 \ncatalyze the removal of acetyl groups from histones, thereby promoting chromatin 219 \ncondensation and transcriptional repression. To investigate the effect of HSV -1 220 \ninfection on HDAC expression and histone acetylation, we exam ined the protein 221 \nlevels of key HDAC isoforms following viral infection. Immunoblotting analysis  222 \nrevealed a marked decrease in HDAC1 and HDAC2 protein levels in cells infected 223 \nwith either HSV -1 or PRV , whereas the expression of HDAC4, HDAC6, and 224 \nHDAC11 remained unchanged (Fig. 1A, B). In contrast, qRT -PCR results showed no 225 \nalterations in HDAC1 and HDAC2 mRNA levels (Fig. 1C, D), suggesting that their 226 \ndepletion is regulated at the post-translational level. 227 \nTo assess the functional impact of HDAC1/2 reduction,  we examined global histone 228 \nacetylation patterns. Site -specific immunoblotting demonstrated elevated acetylation 229 \nat multiple lysine residues, including H3K9, H3K27, H3K56, H4K8, and H4K12, in 230 \nHSV-1-infected HeLa cells (Fig. 1E). A similar hyperacetylation profile was observed 231 \nin PRV-infected 3D4/21 cells and in murine liver tissues infected with HSV-1 (Fig. 1F, 232 \nG). Together, these findings indicate that HSV-1 selectively degrades class I HDACs, 233 \nresulting in widespread histone hyperacetylation that fosters a  chromatin state 234 \nconducive to viral replication. 235 \nHSV-1 infection induces DDR through HDAC1/2 depletion and histone 236 \nhyperacetylation. 237 \nGiven that excessive histone acetylation can destabilize chromatin structure, we 238 \nsought to elucidate the mechanism by which this epigenetic modification activates the 239 \nDDR pathway. Accumulating evidence underscores the pivotal role of histone 240 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nmodifications in maintaining genomic stability, with dynamic changes in chromatin 241 \narchitecture being intimately associated with DDR initiation. To determine whether 242 \nHSV-1 infection induces DDR activation, we evaluated the phosphorylation of 243 \nγ-H2AX, a well -established ma rker of DNA double -strand breaks. 244 \nImmunofluorescence microscopy revealed a significant increase in γ -H2AX foci in 245 \ncells infected with HSV -1 or PRV (Fig. 2A, B). Comet assays further corroborated 246 \nenhanced DNA fragmentation under these conditions (Fig. 2C –F). Given the central 247 \nrole of ATM/ATR signaling in the DDR, we proceeded to examine the activation of 248 \nkey checkpoint kinases. Immunoblotting analysis confirmed phosphorylation of ATM, 249 \nATR, Chk1, Chk2, H2AX, and RAD51 in HSV -1-infected HeLa cells, 3D4/21 cell s, 250 \nand murine liver tissues (Fig. 2G, H). 251 \nMoreover, pharmacological inhibition of ATR using berzosertib markedly reduced 252 \nviral titers in plaque assays (Fig. 2I). In vivo , administration of berzosertib 253 \nsignificantly improved survival rates in HSV -1-infected mice (Fig. 2J). These results 254 \ndemonstrate that HSV -1 infection activates the DDR, likely mediated through 255 \nHDAC1/2 degradation and consequent histone hyperacetylation. This virus -induced 256 \nDDR activation underscores the profound impact of HSV -1 on host chrom atin 257 \ndynamics and genomic integrity. 258 \n 259 \nHSV-1 promotes the ubiquitin-proteasome degradation of HDAC1/2. 260 \nTo elucidate the mechanism responsible for HDAC1/2 degradation, we focused on 261 \ntheir post -translational regulation. Since HDAC1/2 mRNA levels were unchanged 262 \nafter infection, we hypothesized that the reduction in HDAC1/2 protein is mediated 263 \nthrough enhanced proteolytic degradation. To identify the relevant degradation 264 \npathway, HSV-1-infected HeLa cells were treated with either the proteasome inhibitor  265 \nMG-132 or autophagy inhibitors (3 -MA and chloroquine). Immunoblot ting analysis 266 \nindicated that only MG -132 rescued HDAC1/2 protein levels (Fig. 3A), confirming 267 \nthat degradation occurs primarily via the proteasomal pathway. 268 \nTo examine whether HSV-1 infection induced ubiquitination of HDAC1, endogenous 269 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nimmunoprecipitation assays were performed, which revealed increased ubiquitination 270 \nof HDAC1 following infection (Fig. 3B). Subsequent ubiquitination assays using 271 \nwild-type (WT), K48 -only, and K63 -only ubiquitin  mutants demonstrated that 272 \nHDAC1 undergoes K63 -linked—but not K48 -linked—polyubiquitination (Fig. 3C). 273 \nUsing a series of HDAC1 truncation mutants, we mapped the ubiquitination site to the 274 \nN-terminal region (amino acids 1–100) (Fig. 3D–F). Site-directed mutagenesis further 275 \nidentified lysine 74 (K74) as the critical residue required for ubiquitination (Fig. 3G –276 \nI). Sequence alignment showed that HDAC2 K75 corresponds to HDAC1 K74 (Fig. 277 \n3J), and a K75R mutation in HDAC2 similarly abolished ubiquitination (Fig. 3J). 278 \nTogether, these results indicate that HSV -1 infection promotes K63 -linked 279 \npolyubiquitination of HDAC1/2 at conserved lysine residues, ultimately leading to 280 \ntheir proteasomal degradation.  281 \n 282 \nMDM2 functions as the E3 ligase mediating HDAC1/2 ubiquitination and 283 \ndegradation. 284 \nHaving established the occurrence of K63 -linked ubiquitination (Fig. 3), we next 285 \nsought to identify the E3 ubiquitin ligase responsible. An RNAi screen targeting 286 \ncandidate E3 ligases revealed MDM2 as the principal mediator of HDAC1/2 287 \ndegradation. Knockdown of MDM2 —but not of PIRH2, KCTD11, CHFR, UHRF1, 288 \nor TRIM46 —effectively prevented HDAC1/2 depletion (Fig. 4A, B). Co -IP assay 289 \nfurther confirmed an enhanced interaction between HDAC1 and MDM2 following 290 \nHSV-1 infection (Fig. 4C). 291 \nTo characterize this interaction, we focused on the RING finger domain of MDM2, 292 \nwhich is essential for its E3 ligase activity. Co -IP analysis demonstrated that HSV -1 293 \ninfection promoted binding between FLAG-HDAC1 and EGFP-MDM2, but not with 294 \na RING -deleted mutant (E GFP-MDM2∆RING) (Fig. 4D), indicating that the 295 \ninteraction was RING domain -dependent. To identify the region within HDAC1 296 \nrequired for binding, HeLa cells were co -transfected with EGFP -MDM2 and either 297 \nFLAG-HDAC1 or the ubiquitination -resistant mutant FLAG -HDAC1 K74R. 298 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nInfection with HSV-1 failed to induce binding between EGFP -MDM2 and the K74R 299 \nmutant (Fig. 4E), underscoring the essential role of lysine 74 in mediating 300 \nMDM2-dependent ubiquitination. 301 \nUbiquitination assay further revealed that overexpression of EGFP-MDM2∆RING did 302 \nnot enhance HSV -1-induced ubiquitination or degradation of HDAC1 (Fig. 4F). 303 \nSimilarly, the FLAG-HDAC1 K74R mutant remained resistant to ubiquitination under 304 \nall experimental conditions (Fig. 4F). Collectively, these results demonstrate t hat 305 \nHSV-1 induces HDAC1/2 degradation through MDM2 -mediated K63 -linked 306 \nubiquitination. 307 \n 308 \nHSV-1 triggers cytoplasmic translocation of HDAC1 to facilitate its degradation. 309 \nTo elucidate the mechanisms by which HSV -1 induces HDAC degradation, we 310 \nsystematically analyzed the subcellular localization of HDAC1. In mock -infected 311 \nHeLa cells, HDAC1 was predominantly nuclear; however, HSV -1 infection triggered 312 \na pronounced translocation of HDAC1 to the cytoplasm (Fig. 5A). This redistribution 313 \nwas completely abolished by treatment with leptomycin B (LMB), a specific inhibitor 314 \nof CRM1-mediated nuclear export (Fig. 5A). 315 \nTo determine whether translocation is functionally linked to degradation, we 316 \nconducted cycloheximide (CHX) chase assays. Degradation of both HDAC1 and 317 \nHDAC2 began approximately 12 hours after CHX treatment and was markedly 318 \naccelerated by HSV -1 infection (Fig. 5B). Importantly, LMB treatment restored 319 \ndegradation kinetics to levels comparable to those in mock -infected cells (Fig. 5B). 320 \nMoreover, under LMB treatm ent, HSV -1 was unable to induce ubiquitination of 321 \nendogenous HDAC1 (Fig. 5C). 322 \nImmunoblotting analysis further indicated that LMB suppressed HSV -1–induced 323 \nphosphorylation of p53 and H2AX (Fig. 5E), confirming that CRM1 -dependent 324 \nnuclear export is essential for MDM2-mediated HDAC1 degradation. Notably, LMB 325 \ntreatment—which inhibits CRM1 -dependent export and consequently prevents 326 \nHDAC1 degradation —significantly impaired viral replication (Fig. 5F). Together, 327 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nthese findings demonstrate that HSV -1 infection promo tes cytoplasmic translocation 328 \nof HDAC1, facilitating its ubiquitination and subsequent proteasomal degradation.  329 \n 330 \nDiscussion 331 \nHSV-1 manipulates host cellular pathways to facilitate its replication, latency, and 332 \nreactivation [2]. In this study, we demonstrate that alphaherpesvirus  targets class I 333 \nhistone deacetylases HDAC1 and HDAC2 for MDM2 -mediated K63 -linked 334 \npolyubiquitination and subsequent proteasomal degradation. This degradation induces 335 \nhistone hyperacetylation, chromatin relaxation, and enhanced viral 336 \nreplication—revealing a mechanism through which alphaherpesvirus reprograms the 337 \nhost epigenetic landscape to optimize viral replication (Fig. 6). 338 \nUnlike human cytomegalovirus, which upregulates HDAC expression to suppress 339 \nhost immunity  [24], HSV-1 promotes the degradation of HDAC1/2 to establish a 340 \nchromatin environment conducive to viral transcription. This highlights a fundamental 341 \ndivergence in epigenetic strategies among herpesviruses. Our findings indicate that 342 \nvirus-directed degradation of HDAC1/2 occurs specifically during lytic replication  343 \n[25], suggesting a dual role for these deacetylases: their presence supports latency, 344 \nwhereas their degradation facilitates lytic replication.  345 \nWe identified MDM2  [26] as the principal E3 ubiquitin ligase responsible for 346 \nubiquitinating HDAC1 at lysine 74 and HDAC2 at lysine 75, leading to their 347 \nproteasomal degradation. This mechanism is distinct from other E3 ligases, including 348 \nPIRH2 [27] and TRIM46 [28]), and critically depends on the RING domain of MDM2. 349 \nWhile K63-linked ubiquitination is typically associated with non-proteolytic functions, 350 \nour work establishes its novel role in mediating HDAC1/2 degradation—uncovering a 351 \npreviously unrecognized viral pathogenesis pathway. Targeting this axis using MDM2 352 \ninhibitors or inhibiting nuclear export of HDAC1/2 significantly restricts viral 353 \nreplication, suggesting that host -directed antiviral strategies may help overcome 354 \nresistance to direct-acting agents. 355 \nDepletion of HDAC1/2 increases acet ylation at histone residues associated with open 356 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nchromatin and active transcription. Similar chromatin remodeling has been observed 357 \nin other viral infections  [29], underscoring its conserved role in viral gene regulation. 358 \nOur results are consistent across multiple cell types, including HeLa, 3D4/21, and 359 \nmurine liver, emphasizing the central importance of HDAC1/2 suppression in HSV -1 360 \nbiology. Notably, HDAC1/2 degradation activates DDR, as indicated by increased 361 \nγ-H2AX foci and phosphorylation of ATM, ATR, Chk1, and Chk2. This aligns with 362 \nprevious reports that HSV -1 exploits D DR signaling to enhance replication  [30-32]. 363 \nImportantly, HDAC1/2 undergo CRM1 -dependent nuclear ex port prior to 364 \ndegradation—a process inhibited by leptomycin B —highlighting the essential role of 365 \nsubcellular localization in viral manipulation of host proteins. 366 \nHSV-1–mediated HDAC1/2 degradation likely supports viral replication through 367 \nmultiple mechanisms, including evasion of nuclear antiviral sensors and stimulation 368 \nof viral gene expression. Targeting the MDM2–HDAC1/2 axis represents a promising 369 \nantiviral strategy. MDM2 inhibitors reduce HSV -1 titers  [33] , suggesting that 370 \nmodulating ubiquitin ation pathways could complement existing therapies. Beyond 371 \nherpesviruses, the MDM2–HDAC axis may be exploited by other DNA viruses (e.g., 372 \nHBV , HPV) that manipulate chromatin and DDR, indicating a possible common host 373 \nvulnerability. However, broad HDAC inhi bition may unintentionally enhance viral 374 \nreplication, underscoring the need for targeted therapeutic approaches. 375 \nThis study primarily employed in vitro models (e.g., HeLa and 3D4/21 cells), which 376 \nlack the neuronal environment essential for studying HSV -1 l atency. Future work 377 \nshould validate these findings in primary neurons and trigeminal ganglia  [34]. 378 \nAdditional studies are also needed to determine whether HDAC1/2 degradation 379 \noccurs during reactivation or is limited to lytic infection  [8], and to explore the 380 \ninterplay between HDAC degradation, viral tegument proteins, and host stress 381 \nresponses. In vivo evaluation of inhibitors targeting the MDM2/HDAC1 –2 pathway 382 \nwill be crucial for assessing their therapeutic potential. 383 \n 384 \nAuthor contributions 385 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nSheng-Li Ming  and Meng-Hua Du  : Data curation, Formal analysis, Investigation, 386 \nMethodology, Validation, Visualization, Writing – original draft; Jia -Ming Yang: Data 387 \ncuration, Formal analysis, Investigation, Validation; Ya -Di Guo: Data curation, Formal 388 \nanalysis, Investigation, Validation, Visualization; Jia-Jia Pan: Data curation, Formal analysis, 389 \nInvestigation, Methodology, Visualization; Wei -Fei Lu: Data curation, Formal analysis, 390 \nInvestigation; Jiang Wang: Methodology, Visualization, Project administration, Supervision, 391 \nWriting – review & editing; Lei Zeng and Bei -Bei Chu: Conceptualization, Data curation, 392 \nFunding acquisi -tion, Investigation, Methodology, Project administration, Resources, 393 \nSupervision, Validation, Writing – original draft, Writing – review & editing. 394 \n 395 \nDeclaration of competing interest 396 \nThe authors declare that they have no competing interests. 397 \n 398 \nEthics statement 399 \nAll procedures involving animals in this study received ethical approval from the 400 \nInstitutional Animal Care and Use Committee (IACUC) of Henan Agricultural 401 \nUniversity (Permit Number: HNND20 20031008). This research was performed in 402 \naccordance with the regulations specified in the Guide for the Care and Use of 403 \nLaboratory Animals established by the Ministry of Science and Technology of China 404 \n 405 \nFunding 406 \nThis research was supported by grants from the National Key R&D Program of China 407 \n(2021YFD1301200 and 2023YFD1801600), the China Postdoctoral Science 408 \nFoundation (2025M770267, GZC20240430). 409 \n 410 \nData Availability Statement 411 \nThe datas that support the findings of this study are openlyavailable in “Mendeley Data , V3” 412 \nat https://data.mendeley.com/datasets/yg5fgtvxzk/3, doi: 10.17632/yg5fgtvxzk.3, reference 413 \nnumber[35]. 414 \n 415 \nDeclaration of generative AI and AI-assisted technologies in the writing process 416 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nThis study affirms that neither generative artificial intelligence nor any other 417 \nAI-assisted technologies were employed in the preparation of this manuscript. 418 \n 419 \nReferences 420 \n1. Mohammed Tanveer H, Brent A S, David I B. Small Animal Models to Study Herpes Simplex Virus 421 \nInfections. Viruses. 2024;16(7). doi: 10.3390/v16071037. PubMed PMID: 39066200. 422 \n2. Maria K. HSV-1 virions and related particles: biogenesis and implications in the infection. J Virol. 423 \n2025;99(3). doi: 10.1128/jvi.01076-24. PubMed PMID: 39898651. 424 \n3. Derek G, Daniel P D, Carol A H, Moriah L S, Paola K V, Mária B, et al. ICTV Virus Taxonomy Profile: 425 \nHerpesviridae 2021. 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It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nPubMed PMID: 24850735. 504 \n32. Govind A S, Clodagh C OS. Viral and Cellular Genomes Activate Distinct DNA Damage Responses. 505 \nCell. 2015;162(5). doi: 10.1016/j.cell.2015.07.058. PubMed PMID: 26317467. 506 \n33. Shengli M, Shijun Z, Jiayou X, Guoyu Y , Lei Z, Jiang W, et al. Alphaherpesvirus manipulates retinoic 507 \nacid metabolism for optimal replication. iScience. 2024;27(7). doi: 10.1016/j.isci.2024.110144. 508 \nPubMed PMID: 38989466. 509 \n34. Yue D, Yuqi L, Siyu C, Yuhang X, Hongjia C, Shuyuan Q, et al. Neuronal miR -9 promotes HSV -1 510 \nepigenetic s ilencing and latency by repressing Oct -1 and Onecut family genes. Nat Commun. 511 \n2024;15(1). doi: 10.1038/s41467-024-46057-6. PubMed PMID: 38443365. 512 \n35. Sheng-Li Ming M -HD, Jia- Ming Yang, Ya -Di Guo, Jia -Jia Pan, Wei -Fei Lu, Jiang Wang, Lei Zeng, 513 \nBei-Bei Chu. The targeted cytosolic degradation of class I histone deacetylases is essential for efficient 514 \nalphaherpesvirus replication Mendeley Data. 2025. doi: 10.17632/yg5fgtvxzk.3. 515 \n 516 \n  517 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\nFigure legends 518 \nFigure 1. Viral infection induces degradation of class I HDACs  and 519 \nhyperacetylation of histones H3 and H4. 520 \n(A) Immunoblotting analysis  of the indicated HDACs in HeLa cells infected with 521 \nHSV-1 (MOI = 1) for the indicated time points. 522 \n(B) Immunoblotting analysis of the indicated HDACs in 3D4/21 cells infected with 523 \nPRV-QXX (MOI = 1) for the indicated time points. 524 \n(C and D) qRT-PCR analysis of HDAC1 and HDAC2 mRNA levels in HSV-1-infected 525 \nHeLa cells (C) or PRV -QXX-infected 3D4/21 cells (D) (MOI = 1). Data are mean ± 526 \nSEM; n = 3. **P < 0.01, ***P < 0.001. 527 \n(E) Immunoblotting of the indicated proteins in HeLa cells from  infected with HSV-1 528 \n(MOI = 1) for the indicated time points. 529 \n(F) Immunoblotting of the indicated proteins in 3D4/21 cells from infected with 530 \nPRV-QXX (MOI = 1) for the indicated time points.   531 \n(G) Immunoblotting analysis of the indicated proteins in liver tissues from mice 532 \nmock-infected or infected with HSV -1 (1 × 10⁶ pfu per mouse) at 5 days 533 \npost-infection (n = 3).  534 \n 535 \nFigure 2. Viral infection triggers chromatin dysfunction and DDR activation via 536 \nHDAC degradation. 537 \n(A and B ) Immunofluorescence staining of γ -H2AX (red) and H2AX ( green) in 538 \nHSV-1-infected HeLa cells (A) or PRV -infected 3D4/21 cells (B) (MOI = 1). Scale 539 \nbars: 10 μm.   540 \n(C and D ) Comet assay assessing DNA damage in HeLa cells infected with HSV -1 541 \n(MOI = 1). Data are mean ± SEM; n = 40 cells/group. **P < 0.01, ***P < 0.001. 542 \n(E and F ) Comet assay assessing DNA damage in 3D4/21 cells infected with PRV 543 \n(MOI = 1) at the indicated time points. Data are mean ± SEM; n = 40 cells/group. **P 544 \n< 0.01, ***P < 0.001. 545 \n(G and H) Immunoblotting analysis  of DDR markers (p -ATM, p -ATR, p -Chk1, 546 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\np-Chk2, γ-H2AX) and viral ICP4/EP0 in HSV -1-infected HeLa cells (G) or 547 \nPRV-QXX-infected 3D4/21 cells (H) (MOI = 1).  548 \n(I) Viral titers in HSV -1-infected HeLa cells (MOI = 1) treated with berzosertib (50 549 \nnM) or vehicle. Data are mean ± SEM; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001  550 \n(J) Survival curves of HSV -1-infected mice (1 × 10⁶ PFU/mouse) treated with 551 \nberzosertib (20 mg/kg) or vehicle (n = 12/group), ***P < 0.001. 552 \n 553 \nFigure 3. Viral infection induces K63-linked ubiquitination of HDAC1/2. 554 \n(A) Immunoblotting analysis of the indicated proteins in HeLa cells either 555 \nmock-infected or infected with HSV -1 (MOI = 1), treated with vehicle, 3 -MA (10 556 \nμM), MG-132 (10 μM), or chloroquine (10 μM) for the indicated times. 557 \n(B) Ubiquitination assay of HDAC1 in HeLa cells eith er mock -infected or infected 558 \nwith HSV-1 (MOI = 1) at 24 hpi. 559 \n(C) Ubiquitination assay of FLAG -HDAC1 in HeLa cells transfected with 560 \nHA-ubiquitin variants (WT, K48, K63), and either mock -infected or infected with 561 \nHSV-1 (MOI = 1) at 24 hpi. 562 \n(D) Schematic representation of HDAC1 deletion mutants. 563 \n(E-I) Ubiquitination assays of FLAG -HDAC1 mutant variants in HeLa cells either 564 \nmock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. 565 \n(J) Ubiquitination assays of FLAG -HDAC2 variants (WT and K75R) in HeLa cells 566 \neither mock-infected or infected with HSV-1 (MOI = 1) at 24 hpi. 567 \n 568 \nFigure 4. MDM2 mediates viral -induced HDAC1/2 degradation via K74/K75 569 \nubiquitination. 570 \n(A) qRT-PCR analysis validation of E3 ligase knockdown efficiency (shPIRH2, 571 \nshKCTD11, shMDM2, shCHFR, shUHRF1, shTRIM46) in HeLa cells. Data are mean 572 \n± SEM; n = 3. ***P < 0.001. 573 \n(B) Immunoblotting analysis  of HDAC1/2 stability in E3 ligase -knockdown HeLa 574 \ncells infected with HSV-1 (MOI = 1). 575 \n(C) Co-IP assay of endogenous HDAC1 -MDM2 interaction in HSV -1-infected HeLa 576 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\ncells (MOI = 1; 24 hpi).   577 \n(D) Co-IP assay of FLAG -HDAC1 with EGFP -MDM2 (WT or ΔRING) in 578 \nHSV-1-infected HeLa cells (MOI = 1; 24 hpi). 579 \n(E) Co-IP assay of FLAG -HDAC1 (WT or K74R) with EGFP -MDM2 in 580 \nHSV-1-infected HeLa cells (MOI = 1; 24 hpi).   581 \n(F) Ubiquitination assays of FLAG -HDAC1 (WT or K74R) co -expressed with 582 \nEGFP-MDM2 (WT or ΔRING) in HSV-1-infected HeLa cells (MOI = 1; 24 hpi).  583 \n 584 \nFigure 5. Cytoplasmic translocation enables MDM2 -mediated HDAC1 585 \ndegradation. 586 \n(A) Immunofluorescence staining of HDAC1 ( red) and viral ICP4 ( green) in 587 \nHSV-1-infected HeLa cells (MOI = 1) ± leptomycin B (LMB; 10 nM; 24 h). Scale bar: 588 \n10 μm. 589 \n(B) CHX chase assay measuring HDAC1/2 degradation kinetics in HSV -1-infected 590 \nHeLa cells (MOI = 1) ± LMB (10 μM).  591 \n(C) Ubiquitination assay of HDAC1 in HSV-1-infected HeLa cells (MOI = 1) ± LMB 592 \n(10 μM; 24 h). 593 \n(D) Immunofluorescence analysis of MDM2 (red) and ICP4 (green) in 594 \nHSV-1-infected HeLa cells (MOI = 1) ± LMB (10 μM; 24 h). Scale bar: 10 μm. 595 \n(E) Immunoblotting analysis  of DDR markers (γ -H2AX) and HDAC1/2 in 596 \nHSV-1-infected HeLa cells (MOI = 1) ± LMB (10 μM). 597 \n(F) Viral titers in HSV -1-infected HeLa cells (MOI = 1) ± LMB (10 μM). Data are 598 \nmean ± SEM; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001  599 \n 600 \nFigure 6. A schematic model showing the targeted cytosolic degradation of class I 601 \nhistone deacetylases is essential for efficient alphaherpesvirus replication. 602 \n 603 \n 604 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2026.01.02.697337doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}