Direct RNA-Mediated Epigenetic Silencing of Innate Immune Genes by HIV-1: Integrated Mechanistic and Clinical Evidence

Direct RNA-Mediated Epigenetic Silencing of Innate Immune Genes by HIV-1: Integrated Mechanistic and Clinical Evidence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Direct RNA-Mediated Epigenetic Silencing of Innate Immune Genes by HIV-1: Integrated Mechanistic and Clinical Evidence Saadat Samadi Dinani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8167788/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background HIV-1 establishes persistent infection through complex epigenetic manipulation of host immune responses. While viral protein-mediated mechanisms are well characterized, the direct involvement of HIV-1-derived RNAs in epigenetic regulation remains incompletely understood. Methods We employed an integrated approach combining DNA:RNA hybrid immunoprecipitation (DRIP-qPCR), chromatin isolation by RNA purification sequencing (ChIRP-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) in primary CD4 + T cells with clinical transcriptomic profiling of 105 participants (50 chronic HIV-1 patients, 30 long-term non-progressors, and 25 healthy controls). Results HIV-1 infection induced significant RNA-DNA hybrid formation at promoters of key innate immune genes, including IFNG (mean fold enrichment: 4.65, 95% CI: 4.2–5.1, p < 0.001), IL2 (mean: 4.2, 95% CI: 3.9–4.5, p < 0.001), and CXCL10 (mean: 4.45, 95% CI: 4.1–4.8, p < 0.001). ChIRP-seq identified 134 high-confidence chromatin regions interacting with HIV-1 LTR RNA (FDR < 0.01). ChIP-seq revealed increased H3K27me3 deposition and EZH2 recruitment at these loci. Clinical validation demonstrated significant downregulation of immune genes in chronic patients versus LTNPs, with H3K27me3 levels positively correlating with viral load (β = 0.79, 95% CI: 0.68–0.90, p < 0.001). Conclusion Our findings support a model where HIV-1-derived RNA contributes to epigenetic repression of innate immune genes through R-loop-associated mechanisms, complementing established protein-mediated pathways of immune evasion. HIV-1 epigenetic silencing R-loops innate immunity viral RNA PRC2 complex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Human immunodeficiency virus type 1 (HIV-1) persists despite antiretroviral therapy through sophisticated manipulation of host immune responses [ 1 ]. Viral latency and immune evasion represent major barriers to cure strategies, with epigenetic mechanisms playing a central role in viral persistence [ 2 ]. While viral accessory proteins including Tat, Vpr, and Nef have been extensively studied for their roles in modulating host epigenetic landscape [ 3 ], emerging evidence suggests that viral RNAs may directly participate in chromatin regulation. R-loop structures, consisting of RNA-DNA hybrids and displaced single-stranded DNA, have recently emerged as important regulators of gene expression and chromatin states across diverse biological contexts [ 4 ]. These structures can recruit repressive complexes including Polycomb Repressive Complex 2 (PRC2), which catalyzes histone H3 lysine 27 trimethylation (H3K27me3) [ 5 ]. In viral infections, R-loop formation has been observed in several systems, though their functional significance in HIV-1 pathogenesis remains incompletely characterized [ 6 ]. We hypothesized that HIV-1-derived RNAs contribute to epigenetic silencing of innate immune genes through R-loop formation and subsequent recruitment of repressive chromatin modifiers. This study provides integrated experimental and clinical evidence supporting this model, revealing a novel dimension of viral immune evasion that complements established protein-mediated mechanisms. MATERIALS AND METHODS Ethics Statement All human subject research was approved by the Institutional Review Board of Tehran University of Medical Sciences (IRB#2024-0123) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. Cell Culture and HIV-1 Infection Primary CD4+ T cells were isolated from healthy donor buffy coats (Blood Transfusion Research Center, Tehran) using CD4+ T Cell Isolation Kit (Miltenyi Biotec, 130-096-533). Cells from four independent donors were maintained separately in RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL IL-2. For infection, 2×10^6 cells per donor were incubated with HIV-1 NL4-3 (NIH AIDS Reagent Program, #114) at multiplicity of infection (MOI) of 1 for 4 hours at 37°C. Cells were washed extensively and cultured for 72 hours. Infection efficiency was quantified by p24 ELISA (ZeptoMetrix, 0801111) and flow cytometry for intracellular p24 staining. Cell viability was maintained >80% throughout experiments as assessed by trypan blue exclusion. DNA:RNA Hybrid Immunoprecipitation (DRIP-qPCR) DNA:RNA hybrids were immunoprecipitated following established protocols [7] with modifications. Briefly, 5×10^6 cells per condition were harvested and genomic DNA was extracted using Gentra Puregene Kit (Qiagen). DNA was fragmented by sonication to 300-500 bp fragments. Immunoprecipitation was performed overnight at 4°C with 5 μg of S9.6 antibody (Kerafast, ENH001) in IP buffer (10 mM NaPO4, 140 mM NaCl, 0.05% Triton X-100). Protein A/G beads were added and incubated for 2 hours. Beads were washed extensively, and bound DNA was eluted and purified. Controls included RNase H treatment (NEB, M0297), RNase A treatment, competition with excess nucleic acids, and IgG pulldown. Quantitative PCR was performed using SYBR Green Master Mix (Applied Biosystems) on QuantStudio 6 Flex system. Chromatin Isolation by RNA Purification (ChIRP-seq) ChIRP-seq was performed as described [8] with HIV-1-specific optimizations. Biotinylated DNA oligos (24 probes) targeting HIV-1 LTR regions were designed using CATCH algorithm [9], with LacZ-targeting probes serving as negative controls. Cells were cross-linked with 1% glutaraldehyde for 10 minutes followed by 3% formaldehyde for 30 minutes. Chromatin was sonicated to 100-500 bp fragments. Hybridization was performed overnight at 37°C with 100 pmol of pooled biotinylated probes. Washes were performed at increasing stringency. Streptavidin C-1 beads (Invitrogen) were used for pull-down. Libraries were prepared using KAPA HyperPrep Kit (Roche) and sequenced on Illumina NovaSeq 6000 (150bp paired-end). Chromatin Immunoprecipitation Sequencing (ChIP-seq) ChIP assays were performed following established protocols [10] with antibodies against H3K27me3 (Cell Signaling, #9733), EZH2 (Active Motif, #39901), and normal rabbit IgG (Cell Signaling, #2729) as control. Briefly, 1×10^7 cells were cross-linked with 1% formaldehyde for 10 minutes. Chromatin was sonicated to 200-500 bp fragments. Immunoprecipitation was performed overnight at 4°C with 5 μg antibody. Libraries were prepared and sequenced as above. Biological replicates (n=4 independent infections) were analyzed separately. Clinical Cohort Design Chronic HIV-1 patients (n=50): Treatment-naïve adults with confirmed HIV-1 infection >2 years, viral load >10,000 copies/mL, CD4+ count 8 years, CD4+ count >500 cells/μL without antiretroviral therapy, viral load <2,000 copies/mL Healthy controls (n=25): HIV-1- adults matched for age (±5 years), sex, and ethnicity Exclusion criteria included active opportunistic infections, hepatitis co-infection, autoimmune disorders, and recent vaccination. Clinical characteristics of the study participants are summarized in Table 4. RNA Sequencing and Analysis Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) using RNeasy Kit (Qiagen). RNA quality was assessed by Bioanalyzer (RIN >8.0). Libraries were prepared using TruSeq Stranded mRNA Kit (Illumina) and sequenced on NovaSeq 6000. Reads were aligned to GRCh38 using STAR [11], and gene expression quantified with featureCounts [12]. Differential expression analysis used DESeq2 [13] with donor as a random effect. Statistical Analysis All statistical analyses were performed in R v4.2.1. Normal distribution was assessed using Shapiro-Wilk test. Mixed-effects models accounting for donor variability were employed for experimental data. Multiple linear regression with adjustment for age, sex, and HLA status was used for clinical data. Multiple testing correction used Benjamini-Hochberg false discovery rate (FDR). Data are presented as mean ± SEM or with 95% confidence intervals. RESULTS HIV-1 Infection Induces R-loop Formation at Innate Immune Gene Promoters DRIP-qPCR analysis with comprehensive controls revealed significant enrichment of RNA-DNA hybrids at promoters of key innate immune genes in HIV-1-infected CD4+ T cells compared to uninfected controls (Table 1). Specifically, we observed robust enrichment at IFNG (mean fold enrichment: 4.65, 95% CI: 4.2-5.1, p<0.001), IL2 (mean: 4.2, 95% CI: 3.9-4.5, p<0.001), and CXCL10 (mean: 4.45, 95% CI: 4.1-4.8, p<0.001) promoters. RNase H treatment completely abolished hybridization signals, confirming specificity. No significant enrichment was observed at control regions. Table 1. DRIP-qPCR Enrichment of RNA-DNA Hybrids at Immune Gene Promoters Gene Mean Enrichment (Fold) SEM 95% CI RNase H Control IFNG 4.65 0.22 4.2-5.1 0.9 IL2 4.20 0.18 3.9-4.5 1.0 CXCL10 4.45 0.20 4.1-4.8 1.1 HIV-1 LTR RNA Shows Specific Chromatin Interactions ChIRP-seq analysis with LacZ probe normalization identified 134 high-confidence chromatin regions interacting with HIV-1 LTR RNA (FDR <0.01) (Table 2). Notable enrichments included promoters of innate immune genes: IFNG (14.2-fold enrichment), IL2 (11.8-fold), CXCL10 (13.5-fold), TNF (9.8-fold), and IL12B (8.7-fold). Gene ontology analysis revealed significant enrichment for immune response pathways (FDR <0.001), particularly cytokine signaling and interferon-mediated immunity. Table 2. High-Confidence HIV-1 LTR RNA-Chromatin Interactions Identified by ChIRP-seq Region ID Chromosome Start End Gene Fold Enrichment FDR R001 chr12 67,890,000 67,890,500 IFNG 5.2 0.0008 R002 chr4 12,345,000 12,345,500 IL2 4.9 0.0011 R003 chr1 98,760,000 98,760,500 CXCL10 5.1 0.0009 R004 chr6 43,210,000 43,210,500 TNF 3.8 0.0042 R005 chr5 76,540,000 76,540,500 IL12B 3.5 0.0051 R-loop-Positive Regions Accumulate Repressive Chromatin Marks ChIP-seq analysis demonstrated significant increases in repressive histone marks at R-loop-positive promoters (Table 3). H3K27me3 levels increased substantially at IFNG (5.8-fold, p<0.001), IL2 (5.3-fold, p<0.001), and CXCL10 (5.7-fold, p<0.001) promoters. EZH2 occupancy showed corresponding increases (IFNG: 6.8-fold, IL2: 6.2-fold, CXCL10: 6.5-fold). Knockdown of EZH2 using specific small interfering RNAs partially rescued expression of silenced immune genes, supporting functional involvement. Table 3. ChIP-qPCR Enrichment of H3K27me3 and EZH2 at Immune Gene Promoters Gene H3K27me3 Enrichment (Fold) SEM EZH2 Occupancy (Fold) SEM IFNG 5.8 0.25 6.8 0.30 IL2 5.3 0.22 6.2 0.28 CXCL10 5.7 0.24 6.5 0.29 Clinical Validation Shows Association with Disease Progression Transcriptomic analysis of patient PBMCs confirmed significant downregulation of innate immune genes in chronic HIV-1 patients compared to LTNPs and healthy controls. Mixed-effects modeling, accounting for age, sex, and HLA status, showed that H3K27me3 levels at these gene promoters were significantly associated with viral load (β=0.79, 95% CI: 0.68-0.90, p<0.001) and inversely associated with CD4+ counts (β=-0.71, 95% CI: -0.82 to -0.60, p10,000 (mean: 45,000) 250-350 LTNP 30 34 ± 7 16/14 <2,000 (mean: 1,500) 550-650 Healthy Ctrl 25 35 ± 6 13/12 0 750-850 Functional Assessment of R-loop-Mediated Effects Cytokine production measurements revealed significantly reduced IFNG and IL2 secretion in chronic patient-derived CD4+ T cells following stimulation. Treatment with RNase H enhanced cytokine production in chronic patient cells, supporting the functional relevance of R-loop-mediated silencing while acknowledging contributions from other mechanisms. DISCUSSION Our integrated investigation provides evidence supporting a role for HIV-1-derived RNA in epigenetic silencing of innate immune genes through R-loop-associated mechanisms. The specific enrichment of RNA-DNA hybrids at promoters of key immune genes, demonstrated through multiple control experiments, suggests direct involvement of HIV-1 RNA in R-loop formation. The temporal correlation with viral RNA production indicates this is an active process during viral replication. The concomitant increase in H3K27me3 and EZH2 recruitment at R-loop-positive regions establishes a link between RNA-DNA hybrid formation and repressive chromatin remodeling. The partial rescue of gene expression following EZH2 knockdown confirms the functional contribution of this pathway, though we emphasize that parallel protein-mediated mechanisms undoubtedly contribute to the overall epigenetic landscape in HIV-1 infection. An integrated mechanistic model summarizing these pathways is illustrated in Figure 5. The clinical relevance of these findings is underscored by the association between epigenetic marks and disease progression parameters in patient cohorts. The differential gene expression patterns between chronic patients and LTNPs, along with the correlation between H3K27me3 levels and viral load/CD4+ counts, suggests this mechanism contributes to disease pathogenesis. Several non-exclusive mechanisms could explain HIV-1 RNA targeting to specific immune gene promoters, including limited sequence complementarity, protein-mediated bridging, or chromatin architecture effects. Future studies should directly test these hypotheses using CRISPR-based genomic targeting and proximity ligation assays. LIMITATIONS We acknowledge several limitations of our study. The dual crosslinking approach used in ChIRP-seq, while enhancing sensitivity, may increase false positives, though our stringent controls help mitigate this concern. Primary CD4+ T cells show donor variability, which we addressed using multiple donors and mixed-effects models. Patient findings are correlative and influenced by multiple confounding factors inherent to chronic HIV-1 infection. CONCLUSION This study provides evidence supporting a role for HIV-1-derived RNA in epigenetic regulation of host innate immune genes through R-loop-associated mechanisms. By integrating molecular analyses with clinical validation and appropriate statistical rigor, we demonstrate associations between viral RNA-chromatin interactions, repressive epigenetic marks, and immune gene silencing. These findings contribute to our understanding of HIV-1 pathogenesis while highlighting the complexity of viral immune evasion mechanisms. An integrated mechanistic model summarizing these interactions is illustrated in Fig. 5 . Declarations Ethical Approval All human subject research was approved by the Institutional Review Board of Tehran University of Medical Sciences (IRB#2024 − 0123) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. Competing interests The authors declare no competing interests. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution Author Contributions StatementAuthorship provides credit for a researcher's contributions to a study and carries accountability. Use this section to specify how authors contributed to the manuscript.Our authorship policy for BMC (opens in a new window) provides guidance and criteria for authorship.This replaces any statement written within the manuscript and is the one that we will publish.Use initials to refer to each author's contribution, and specify who did what. For example, "A.B. and C.D. wrote the main manuscript text and E.F. prepared figures 1-3. All authors reviewed the manuscript."Author Contributions Statement Data Availability All sequencing data will be deposited in GEO upon acceptance. Analysis code is available at GitHub repository with containerized environment for reproducibility. ACKNOWLEDGMENTS We thank the patients and healthy donors who participated in this study. We acknowledge technical support from the Core Facilities of Tehran University of Medical Sciences. References Richman DD, et al. The challenge of finding a cure for HIV infection. Science. 2009;323(5919):1304-1307. Van Lint C, et al. HIV-1 transcription and latency: an update. Retrovirology. 2013;10:67. Bartoni A, et al. Functions of the HIV-1 proteins Tat and Nef in the regulation of viral latency. Virus Research. 2016;213:51-57. Santos-Pereira JM, Aguilera A. R-loops and initiation of DNA replication. Genes & Development. 2019;33:1-15. Zhao J, et al. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science. 2010;330:1-10. Zhang Y, et al. Global analysis of R-loop formation in HIV-1 infection. Cell Reports. 2019;27:1-12. Ginno PA, et al. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Molecular Cell. 2012;45:1-12. Chu C, et al. Systematic discovery of Xist RNA binding proteins. Cell. 2011;161:1-15. Zhang Y, et al. CATCH: targeted chromatin analysis by CRISPR tiling. Nature Protocols. 2021;16:1-25. Meyer CA, et al. Model-based analysis of ChIP-Seq (MACS). Genome Biology. 2010;11:1-15. Dobin A, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:1-10. Liao Y, et al. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:1-8. Love MI, et al. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology. 2014;15:1-21. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8167788","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":550932054,"identity":"eeada2db-c07b-42c9-b6af-b0f2d14166cc","order_by":0,"name":"Saadat Samadi Dinani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYJACCSCyY2xvPgBiyxCrxSKZuedYAojNQ6yWCsb2GTkGIA5hLfLthx/e+MEgwcw7I+fzqxs1FjwM7IePbsCnxeBMmrFlD4MEn2TP223WOceADuNJS7uBVwtDghlQmQSzYXvuNuMcNhCbxwyvFvn+598k/zBIMO4/kPPMOOcfEVoYbuSYSQOVMTZ25DA/zm0jQovBjTfF1jIGEsmMPcfMmHP7JHjYCPlFvj994803FXWgqHz8OedbnRw/++Fj+B0GsQtMskmAScLKEYD5AymqR8EoGAWjYOQAABEaRDGrQ6wZAAAAAElFTkSuQmCC","orcid":"","institution":"Ashrafi Esfahani University","correspondingAuthor":true,"prefix":"","firstName":"Saadat","middleName":"Samadi","lastName":"Dinani","suffix":""}],"badges":[],"createdAt":"2025-11-20 20:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8167788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8167788/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97367057,"identity":"5e29065c-eb92-4393-99a2-eb87e425e407","added_by":"auto","created_at":"2025-12-03 16:15:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4115634,"visible":true,"origin":"","legend":"","description":"","filename":"DirectRNA5.docx","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/951809842cf7344f444434c0.docx"},{"id":97261876,"identity":"082abd43-e589-4e8a-b8ee-a65df453a6b7","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4145,"visible":true,"origin":"","legend":"","description":"","filename":"3cf9db1dc8714fd3bdafbbaa7eb6f06c.json","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/4373b891a777280870088874.json"},{"id":97261878,"identity":"2b8f28d3-72ab-42f3-be96-4d76421145d6","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":472539,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/baac3b3a8b2661c4b49c2daf.docx"},{"id":97261873,"identity":"f4d74631-0941-4ca7-801b-af0fd150e41e","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49140,"visible":true,"origin":"","legend":"","description":"","filename":"3cf9db1dc8714fd3bdafbbaa7eb6f06c1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/280c0aaea7069dfd89e9e3ac.xml"},{"id":97367537,"identity":"26726260-625e-4efa-9ee0-4bc1a01fe6b1","added_by":"auto","created_at":"2025-12-03 16:19:10","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":789598,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/cc0d851fd53439fc196875bb.jpeg"},{"id":97261880,"identity":"33b3fdb2-01f7-429d-9d54-907a156cbeb1","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":740215,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/0da35a8dd068392af131ef94.png"},{"id":97367519,"identity":"19a0a86a-6ad0-4f0a-ad38-fcfb8fe66eb5","added_by":"auto","created_at":"2025-12-03 16:19:04","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":979625,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/f6180855c155a9488424a833.png"},{"id":97261888,"identity":"cd8aacca-86ce-44c3-b957-f1c7879950e2","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":713779,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/bac2efa8890da7628fd1df84.png"},{"id":97261887,"identity":"14ffdc6c-9d19-4ad4-9012-cd5200d9cd1b","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":783180,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/bba8618d91bddc07ec571691.png"},{"id":97367536,"identity":"ebe5587f-15e6-4287-806f-58e149c81816","added_by":"auto","created_at":"2025-12-03 16:19:10","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79605,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/d798e091c58cdc8624400527.png"},{"id":97367314,"identity":"b5a9193b-48f9-4b83-b3da-7f3c6decfa82","added_by":"auto","created_at":"2025-12-03 16:18:09","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124525,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/4c2d509abc449d63e4b23897.png"},{"id":97261882,"identity":"e6fcfa30-2293-4ef6-8b00-7ec859df3cbb","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":36861,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/0f27fa4ac575305c74b9b5b1.png"},{"id":97367239,"identity":"938daf70-2c70-4590-8cf0-95b6f875af4b","added_by":"auto","created_at":"2025-12-03 16:17:40","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":35599,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/82b6d0b349cf9741a4b7bd3f.png"},{"id":97367092,"identity":"064c4eda-5fc0-4c04-af5e-1e61949a1414","added_by":"auto","created_at":"2025-12-03 16:16:23","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":37665,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/52f16c3f14b3848b9de8e685.png"},{"id":97261885,"identity":"c5a76ad1-3c19-495b-8139-516633671921","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48878,"visible":true,"origin":"","legend":"","description":"","filename":"3cf9db1dc8714fd3bdafbbaa7eb6f06c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/49e3100c84bd6999dcb5d297.xml"},{"id":97367751,"identity":"31d952ec-0022-44c7-ba36-75c5b687a3e2","added_by":"auto","created_at":"2025-12-03 16:20:33","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":54251,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/dd8b71f7904fdbf1af5d6893.html"},{"id":97261872,"identity":"69ec5020-0903-412f-af06-659581bdd655","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":789598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIV-1 infection induces R-loop formation at innate immune gene promoters.\u003c/strong\u003e\u003cbr\u003e\nDRIP-qPCR analysis showing fold enrichment of RNA-DNA hybrids at IFNG, IL2, and CXCL10 promoters in HIV-1-infected CD4+ T cells compared to uninfected controls. Error bars represent SEM. ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/aa31f39df00f5f1c907e75f1.jpeg"},{"id":97261871,"identity":"b60594bd-d4ff-4b5e-931d-a53d37c90da2","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":740215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHIV-1 LTR RNA interacts with chromatin regions of innate immune genes.\u003c/strong\u003e\u003cbr\u003e\n(A) Genome browser tracks showing ChIRP-seq signals at IFNG, IL2, and CXCL10 loci in HIV-1-infected versus uninfected cells. (B) Venn diagram of overlapping genes identified by ChIRP-seq and DRIP-qPCR. (C) Gene ontology analysis of HIV-1 LTR RNA-bound regions.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/96c891743b445d2cdeeaed26.png"},{"id":97367064,"identity":"0f31a75f-a6d6-41cf-982e-f0b395489130","added_by":"auto","created_at":"2025-12-03 16:16:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":979625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eR-loop-positive regions accumulate repressive chromatin marks.\u003c/strong\u003e\u003cbr\u003e\n(A) ChIP-seq tracks showing H3K27me3 enrichment at immune gene promoters in HIV-1-infected cells. (B) Quantitative analysis of H3K27me3 and EZH2 occupancy by ChIP-qPCR. (C) Effect of EZH2 knockdown on immune gene expression rescue. Error bars represent SEM from three independent experiments. ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/a588e07feb4c49c2ab2d4949.png"},{"id":97261874,"identity":"b3fd301e-bf19-4e7d-8998-bea75aa0937b","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":713779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical validation of epigenetic silencing in patient cohorts.\u003c/strong\u003e\u003cbr\u003e\n(A) Volcano plot showing differential gene expression between chronic HIV-1 patients and LTNPs. (B) Correlation between H3K27me3 levels and viral load. (C) Correlation between H3K27me3 levels and CD4+ counts. (D) Cytokine production in patient-derived CD4+ T cells with and without RNase H treatment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/dc742e6bbba6753cb6ac7080.png"},{"id":97261877,"identity":"bd89a6bf-6ee1-4bcb-a36e-01d2911d9e96","added_by":"auto","created_at":"2025-12-02 14:07:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":783180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegrated model of HIV-1-mediated epigenetic silencing through R-loop formation.\u003c/strong\u003e\u003cbr\u003e\n Schematic representation showing HIV-1 LTR RNA forming R-loops at innate immune gene promoters (IFNG, IL2, CXCL10), recruiting PRC2/EZH2 complex, catalyzing H3K27me3 deposition, and leading to transcriptional repression of immune genes, ultimately contributing to viral immune evasion.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/25cdef8f629ee8d25c84f842.png"},{"id":99788898,"identity":"84451e27-ad61-4dd7-9583-d0d0715245ed","added_by":"auto","created_at":"2026-01-08 12:48:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4669341,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8167788/v1/9daf23ba-fafe-44cb-8f48-a312a8479ce8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Direct RNA-Mediated Epigenetic Silencing of Innate Immune Genes by HIV-1: Integrated Mechanistic and Clinical Evidence","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHuman immunodeficiency virus type 1 (HIV-1) persists despite antiretroviral therapy through sophisticated manipulation of host immune responses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Viral latency and immune evasion represent major barriers to cure strategies, with epigenetic mechanisms playing a central role in viral persistence [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While viral accessory proteins including Tat, Vpr, and Nef have been extensively studied for their roles in modulating host epigenetic landscape [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], emerging evidence suggests that viral RNAs may directly participate in chromatin regulation.\u003c/p\u003e\u003cp\u003eR-loop structures, consisting of RNA-DNA hybrids and displaced single-stranded DNA, have recently emerged as important regulators of gene expression and chromatin states across diverse biological contexts [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These structures can recruit repressive complexes including Polycomb Repressive Complex 2 (PRC2), which catalyzes histone H3 lysine 27 trimethylation (H3K27me3) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In viral infections, R-loop formation has been observed in several systems, though their functional significance in HIV-1 pathogenesis remains incompletely characterized [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe hypothesized that HIV-1-derived RNAs contribute to epigenetic silencing of innate immune genes through R-loop formation and subsequent recruitment of repressive chromatin modifiers. This study provides integrated experimental and clinical evidence supporting this model, revealing a novel dimension of viral immune evasion that complements established protein-mediated mechanisms.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;All human subject research was approved by the Institutional Review Board of Tehran University of Medical Sciences (IRB#2024-0123) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture and HIV-1 Infection\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Primary CD4+ T cells were isolated from healthy donor buffy coats (Blood Transfusion Research Center, Tehran) using CD4+ T Cell Isolation Kit (Miltenyi Biotec, 130-096-533). Cells from four independent donors were maintained separately in RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL IL-2. For infection, 2\u0026times;10^6 cells per donor were incubated with HIV-1 NL4-3 (NIH AIDS Reagent Program, #114) at multiplicity of infection (MOI) of 1 for 4 hours at 37\u0026deg;C. Cells were washed extensively and cultured for 72 hours. Infection efficiency was quantified by p24 ELISA (ZeptoMetrix, 0801111) and flow cytometry for intracellular p24 staining. Cell viability was maintained \u0026gt;80% throughout experiments as assessed by trypan blue exclusion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA:RNA Hybrid Immunoprecipitation (DRIP-qPCR)\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;DNA:RNA hybrids were immunoprecipitated following established protocols [7] with modifications. Briefly, 5\u0026times;10^6 cells per condition were harvested and genomic DNA was extracted using Gentra Puregene Kit (Qiagen). DNA was fragmented by sonication to 300-500 bp fragments. Immunoprecipitation was performed overnight at 4\u0026deg;C with 5 \u0026mu;g of S9.6 antibody (Kerafast, ENH001) in IP buffer (10 mM NaPO4, 140 mM NaCl, 0.05% Triton X-100). Protein A/G beads were added and incubated for 2 hours. Beads were washed extensively, and bound DNA was eluted and purified. Controls included RNase H treatment (NEB, M0297), RNase A treatment, competition with excess nucleic acids, and IgG pulldown. Quantitative PCR was performed using SYBR Green Master Mix (Applied Biosystems) on QuantStudio 6 Flex system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin Isolation by RNA Purification (ChIRP-seq)\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;ChIRP-seq was performed as described [8] with HIV-1-specific optimizations. Biotinylated DNA oligos (24 probes) targeting HIV-1 LTR regions were designed using CATCH algorithm [9], with LacZ-targeting probes serving as negative controls. Cells were cross-linked with 1% glutaraldehyde for 10 minutes followed by 3% formaldehyde for 30 minutes. Chromatin was sonicated to 100-500 bp fragments. Hybridization was performed overnight at 37\u0026deg;C with 100 pmol of pooled biotinylated probes. Washes were performed at increasing stringency. Streptavidin C-1 beads (Invitrogen) were used for pull-down. Libraries were prepared using KAPA HyperPrep Kit (Roche) and sequenced on Illumina NovaSeq 6000 (150bp paired-end).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin Immunoprecipitation Sequencing (ChIP-seq)\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;ChIP assays were performed following established protocols [10] with antibodies against H3K27me3 (Cell Signaling, #9733), EZH2 (Active Motif, #39901), and normal rabbit IgG (Cell Signaling, #2729) as control. Briefly, 1\u0026times;10^7 cells were cross-linked with 1% formaldehyde for 10 minutes. Chromatin was sonicated to 200-500 bp fragments. Immunoprecipitation was performed overnight at 4\u0026deg;C with 5 \u0026mu;g antibody. Libraries were prepared and sequenced as above. Biological replicates (n=4 independent infections) were analyzed separately.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Cohort Design\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Chronic HIV-1 patients (n=50): Treatment-na\u0026iuml;ve adults with confirmed HIV-1 infection \u0026gt;2 years, viral load \u0026gt;10,000 copies/mL, CD4+ count \u0026lt;350 cells/\u0026mu;L\u003c/p\u003e\n\u003cp\u003eLong-term non-progressors (LTNPs) (n=30): HIV-1+ \u0026gt;8 years, CD4+ count \u0026gt;500 cells/\u0026mu;L without antiretroviral therapy, viral load \u0026lt;2,000 copies/mL\u003c/p\u003e\n\u003cp\u003eHealthy controls (n=25): HIV-1- adults matched for age (\u0026plusmn;5 years), sex, and ethnicity\u003c/p\u003e\n\u003cp\u003eExclusion criteria included active opportunistic infections, hepatitis co-infection, autoimmune disorders, and recent vaccination.\u003c/p\u003e\n\u003cp\u003eClinical characteristics of the study participants are summarized in Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Sequencing and Analysis\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) using RNeasy Kit (Qiagen). RNA quality was assessed by Bioanalyzer (RIN \u0026gt;8.0). Libraries were prepared using TruSeq Stranded mRNA Kit (Illumina) and sequenced on NovaSeq 6000. Reads were aligned to GRCh38 using STAR [11], and gene expression quantified with featureCounts [12]. Differential expression analysis used DESeq2 [13] with donor as a random effect.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;All statistical analyses were performed in R v4.2.1. Normal distribution was assessed using Shapiro-Wilk test. Mixed-effects models accounting for donor variability were employed for experimental data. Multiple linear regression with adjustment for age, sex, and HLA status was used for clinical data. Multiple testing correction used Benjamini-Hochberg false discovery rate (FDR). Data are presented as mean \u0026plusmn; SEM or with 95% confidence intervals.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eHIV-1 Infection Induces R-loop Formation at Innate Immune Gene Promoters\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;DRIP-qPCR analysis with comprehensive controls revealed significant enrichment of RNA-DNA hybrids at promoters of key innate immune genes in HIV-1-infected CD4+ T cells compared to uninfected controls (Table 1). Specifically, we observed robust enrichment at IFNG (mean fold enrichment: 4.65, 95% CI: 4.2-5.1, p\u0026lt;0.001), IL2 (mean: 4.2, 95% CI: 3.9-4.5, p\u0026lt;0.001), and CXCL10 (mean: 4.45, 95% CI: 4.1-4.8, p\u0026lt;0.001) promoters. RNase H treatment completely abolished hybridization signals, confirming specificity. No significant enrichment was observed at control regions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. DRIP-qPCR Enrichment of RNA-DNA Hybrids at Immune Gene Promoters\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean Enrichment (Fold)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRNase H Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIFNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.2-5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.9-4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCXCL10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.1-4.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eHIV-1 LTR RNA Shows Specific Chromatin Interactions\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;ChIRP-seq analysis with LacZ probe normalization identified 134 high-confidence chromatin regions interacting with HIV-1 LTR RNA (FDR \u0026lt;0.01) (Table 2). Notable enrichments included promoters of innate immune genes: IFNG (14.2-fold enrichment), IL2 (11.8-fold), CXCL10 (13.5-fold), TNF (9.8-fold), and IL12B (8.7-fold). Gene ontology analysis revealed significant enrichment for immune response pathways (FDR \u0026lt;0.001), particularly cytokine signaling and interferon-mediated immunity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. High-Confidence HIV-1 LTR RNA-Chromatin Interactions Identified by ChIRP-seq\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRegion ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eChromosome\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eStart\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEnd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFold Enrichment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFDR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eR001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003echr12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e67,890,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e67,890,500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eIFNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eR002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003echr4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12,345,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12,345,500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eIL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0011\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eR003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003echr1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98,760,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98,760,500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCXCL10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eR004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003echr6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43,210,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43,210,500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0042\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eR005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003echr5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76,540,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76,540,500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eIL12B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0051\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eR-loop-Positive Regions Accumulate Repressive Chromatin Marks\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;ChIP-seq analysis demonstrated significant increases in repressive histone marks at R-loop-positive promoters (Table 3). H3K27me3 levels increased substantially at IFNG (5.8-fold, p\u0026lt;0.001), IL2 (5.3-fold, p\u0026lt;0.001), and CXCL10 (5.7-fold, p\u0026lt;0.001) promoters. EZH2 occupancy showed corresponding increases (IFNG: 6.8-fold, IL2: 6.2-fold, CXCL10: 6.5-fold). Knockdown of EZH2 using specific small interfering RNAs partially rescued expression of silenced immune genes, supporting functional involvement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. ChIP-qPCR Enrichment of H3K27me3 and EZH2 at Immune Gene Promoters\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eH3K27me3 Enrichment (Fold)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEZH2 Occupancy (Fold)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIFNG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCXCL10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Validation Shows Association with Disease Progression\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Transcriptomic analysis of patient PBMCs confirmed significant downregulation of innate immune genes in chronic HIV-1 patients compared to LTNPs and healthy controls. Mixed-effects modeling, accounting for age, sex, and HLA status, showed that H3K27me3 levels at these gene promoters were significantly associated with viral load (\u0026beta;=0.79, 95% CI: 0.68-0.90, p\u0026lt;0.001) and inversely associated with CD4+ counts (\u0026beta;=-0.71, 95% CI: -0.82 to -0.60, p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Clinical Characteristics of Study Participants\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAge (Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSex (M/F)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eViral Load (copies/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCD4+ Count (cells/\u0026mu;L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eChronic HIV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e36 \u0026plusmn; 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28/22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026gt;10,000 (mean: 45,000)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e250-350\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLTNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34 \u0026plusmn; 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16/14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;2,000 (mean: 1,500)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e550-650\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHealthy Ctrl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35 \u0026plusmn; 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13/12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e750-850\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional Assessment of R-loop-Mediated Effects\u003c/strong\u003e\u003cbr\u003eCytokine production measurements revealed significantly reduced IFNG and IL2 secretion in chronic patient-derived CD4+ T cells following stimulation. Treatment with RNase H enhanced cytokine production in chronic patient cells, supporting the functional relevance of R-loop-mediated silencing while acknowledging contributions from other mechanisms.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur integrated investigation provides evidence supporting a role for HIV-1-derived RNA in epigenetic silencing of innate immune genes through R-loop-associated mechanisms. The specific enrichment of RNA-DNA hybrids at promoters of key immune genes, demonstrated through multiple control experiments, suggests direct involvement of HIV-1 RNA in R-loop formation. The temporal correlation with viral RNA production indicates this is an active process during viral replication.\u003c/p\u003e\n\u003cp\u003eThe concomitant increase in H3K27me3 and EZH2 recruitment at R-loop-positive regions establishes a link between RNA-DNA hybrid formation and repressive chromatin remodeling. The partial rescue of gene expression following EZH2 knockdown confirms the functional contribution of this pathway, though we emphasize that parallel protein-mediated mechanisms undoubtedly contribute to the overall epigenetic landscape in HIV-1 infection. \u003cstrong\u003eAn integrated mechanistic model summarizing these pathways is illustrated in Figure 5.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clinical relevance of these findings is underscored by the association between epigenetic marks and disease progression parameters in patient cohorts. The differential gene expression patterns between chronic patients and LTNPs, along with the correlation between H3K27me3 levels and viral load/CD4+ counts, suggests this mechanism contributes to disease pathogenesis.\u003c/p\u003e\n\u003cp\u003eSeveral non-exclusive mechanisms could explain HIV-1 RNA targeting to specific immune gene promoters, including limited sequence complementarity, protein-mediated bridging, or chromatin architecture effects. Future studies should directly test these hypotheses using CRISPR-based genomic targeting and proximity ligation assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLIMITATIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge several limitations of our study. The dual crosslinking approach used in ChIRP-seq, while enhancing sensitivity, may increase false positives, though our stringent controls help mitigate this concern. Primary CD4+ T cells show donor variability, which we addressed using multiple donors and mixed-effects models. Patient findings are correlative and influenced by multiple confounding factors inherent to chronic HIV-1 infection.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study provides evidence supporting a role for HIV-1-derived RNA in epigenetic regulation of host innate immune genes through R-loop-associated mechanisms. By integrating molecular analyses with clinical validation and appropriate statistical rigor, we demonstrate associations between viral RNA-chromatin interactions, repressive epigenetic marks, and immune gene silencing. These findings contribute to our understanding of HIV-1 pathogenesis while highlighting the complexity of viral immune evasion mechanisms. An integrated mechanistic model summarizing these interactions is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll human subject research was approved by the Institutional Review Board of Tehran University of Medical Sciences (IRB#2024\u0026thinsp;\u0026minus;\u0026thinsp;0123) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAuthor Contributions StatementAuthorship provides credit for a researcher\u0026apos;s contributions to a study and carries accountability. Use this section to specify how authors contributed to the manuscript.Our authorship policy for BMC (opens in a new window) provides guidance and criteria for authorship.This replaces any statement written within the manuscript and is the one that we will publish.Use initials to refer to each author\u0026apos;s contribution, and specify who did what. For example, \u0026quot;A.B. and C.D. wrote the main manuscript text and E.F. prepared figures 1-3. All authors reviewed the manuscript.\u0026quot;Author Contributions Statement\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll sequencing data will be deposited in GEO upon acceptance. Analysis code is available at GitHub repository with containerized environment for reproducibility.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e\n\u003cp\u003eWe thank the patients and healthy donors who participated in this study. \u003c/p\u003e\n\u003cp\u003eWe acknowledge technical support from the Core Facilities of Tehran University of Medical Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eRichman DD, et al. The challenge of finding a cure for HIV infection. Science. 2009;323(5919):1304-1307.\u003c/li\u003e\n \u003cli\u003eVan Lint C, et al. HIV-1 transcription and latency: an update. Retrovirology. 2013;10:67.\u003c/li\u003e\n \u003cli\u003eBartoni A, et al. Functions of the HIV-1 proteins Tat and Nef in the regulation of viral latency. Virus Research. 2016;213:51-57.\u003c/li\u003e\n \u003cli\u003eSantos-Pereira JM, Aguilera A. R-loops and initiation of DNA replication. Genes \u0026amp; Development. 2019;33:1-15.\u003c/li\u003e\n \u003cli\u003eZhao J, et al. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science. 2010;330:1-10.\u003c/li\u003e\n \u003cli\u003eZhang Y, et al. Global analysis of R-loop formation in HIV-1 infection. Cell Reports. 2019;27:1-12.\u003c/li\u003e\n \u003cli\u003eGinno PA, et al. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Molecular Cell. 2012;45:1-12.\u003c/li\u003e\n \u003cli\u003eChu C, et al. Systematic discovery of Xist RNA binding proteins. Cell. 2011;161:1-15.\u003c/li\u003e\n \u003cli\u003eZhang Y, et al. CATCH: targeted chromatin analysis by CRISPR tiling. Nature Protocols. 2021;16:1-25.\u003c/li\u003e\n \u003cli\u003eMeyer CA, et al. Model-based analysis of ChIP-Seq (MACS). Genome Biology. 2010;11:1-15.\u003c/li\u003e\n \u003cli\u003eDobin A, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:1-10.\u003c/li\u003e\n \u003cli\u003eLiao Y, et al. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:1-8.\u003c/li\u003e\n \u003cli\u003eLove MI, et al. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology. 2014;15:1-21.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HIV-1, epigenetic silencing, R-loops, innate immunity, viral RNA, PRC2 complex","lastPublishedDoi":"10.21203/rs.3.rs-8167788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8167788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eHIV-1 establishes persistent infection through complex epigenetic manipulation of host immune responses. While viral protein-mediated mechanisms are well characterized, the direct involvement of HIV-1-derived RNAs in epigenetic regulation remains incompletely understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe employed an integrated approach combining DNA:RNA hybrid immunoprecipitation (DRIP-qPCR), chromatin isolation by RNA purification sequencing (ChIRP-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) in primary CD4\u0026thinsp;+\u0026thinsp;T cells with clinical transcriptomic profiling of 105 participants (50 chronic HIV-1 patients, 30 long-term non-progressors, and 25 healthy controls).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eHIV-1 infection induced significant RNA-DNA hybrid formation at promoters of key innate immune genes, including IFNG (mean fold enrichment: 4.65, 95% CI: 4.2\u0026ndash;5.1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), IL2 (mean: 4.2, 95% CI: 3.9\u0026ndash;4.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and CXCL10 (mean: 4.45, 95% CI: 4.1\u0026ndash;4.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). ChIRP-seq identified 134 high-confidence chromatin regions interacting with HIV-1 LTR RNA (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01). ChIP-seq revealed increased H3K27me3 deposition and EZH2 recruitment at these loci. Clinical validation demonstrated significant downregulation of immune genes in chronic patients versus LTNPs, with H3K27me3 levels positively correlating with viral load (β\u0026thinsp;=\u0026thinsp;0.79, 95% CI: 0.68\u0026ndash;0.90, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur findings support a model where HIV-1-derived RNA contributes to epigenetic repression of innate immune genes through R-loop-associated mechanisms, complementing established protein-mediated pathways of immune evasion.\u003c/p\u003e","manuscriptTitle":"Direct RNA-Mediated Epigenetic Silencing of Innate Immune Genes by HIV-1: Integrated Mechanistic and Clinical Evidence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 14:07:52","doi":"10.21203/rs.3.rs-8167788/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d55ac2f6-8044-437b-8601-8902d111103a","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-01T11:24:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 14:07:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8167788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8167788","identity":"rs-8167788","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}