CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

immunoprecipitation sequencing (meRIP-seq) to map m6A sites transcriptome wide in primary CD4+ T cells upon knockout of core writers. Results: qPCR interrogation of core m6A machinery in primary CD4+ T cells found that all genes are expressed, and their expression is unaffected by interferon stimulation. Of the 46 genes investigated in the CRISPR-Cas9 knock out system, only the writer complex, the core erasers, and the reader YTHDF2 significantly altered HIV infection. Analysis of the meRIP-seq data for knockouts of m6a writers resulted in a reduction in m6A sites across the transcriptome and lower amounts of m6A deposited on the HIV genome. The average position of m6a peaks over mRNA’s across the human genome was bias towards the 3’ end of the coding sequence. m6A peaks across the HIV genome were clustered at the 3’ end of env and nef across all conditions; and unique peaks were observed in gag and pol in the METTL3 and WTAP knock-out populations. Conclusions: Taken together, these studies provide clarity regarding the role of m6A modification machinery in HIV-1 replication in primary CD4+ T cells. Understanding how these modifications are involved in HIV infection, latency, and pathogenesis may lead to the development of next-generation therapeutics targeting viral RNA. Host Gene SLC35A2 Restricts CXCR4-Tropic HIV-1 Replication at the Stage of Viral Fusion Jamie Guenthoer, Hannah L. Itell, Julie Overbaugh Fred Hutchinson Cancer Center, Seattle, WA, USA Background: Despite the diversity of HIV-1 variants in a chronically infected individual, during transmission, only a few variants seed new infections. These founder viruses predominantly utilize the CCR5 co-receptor to facilitate viral entry into CD4 T cells over the CXCR4 co-receptor. The host factors that contribute to this transmission bottleneck are not well understood. We have recently identified a host gene involved in glycosylation, SLC35A2, that restricts CXCR4-tropic (X4) viruses and enhances CCR5-tropic (R5) virus replication, which recapitulates the selection pattern observed in natural transmission. Preliminary data suggest SLC35A2 acts early in infection, but the exact stage is not known. Methods: SLC35A2 was inactivated by CRISPR-Cas9 in primary CD4 T cells (SLC knockout, KO) and several early steps of viral replication were evaluated: (1) binding of HIV Gag-iGFP to cells by flow cytometry; (2) virus-cell fusion by the BlaM-Vpr fusion assay; (3) production of viral reverse transcription (RT) intermediates measured by qPCR. Unpaired t tests were used to determine significance of SLC KO compared to control cells. Results: SLC35A2 inactivation resulted in an increase in iGFP virus binding of both the X4-tropic NL4-3 and the R5-tropic JRFL viruses as compared to control cells (log2FC = 2, p<0.05). Further, complementing SLC KO cells with exogenous SLC35A2 cDNA reduced binding to wildtype levels, suggesting this effect is specific to SLC35A2. We assessed fusion of the X4-tropic virus LAI and observed a 3.5-fold increase in fusion with SLC KO versus control cells across donors. Finally, after infection with Q23.BG505 (R5) virus, SLC KO cells had significantly reduced production of RT intermediates versus the control (log2FC = -4, p<0.05). Conversely, infecting with LAI (X4) led to an increase in RT intermediates in the SLC KO cells (log2FC = 3, p<0.05) compared to control cells. Conclusions: Overall, these results suggest SLC35A2 acts early in viral replication. SLC35A2’s function alters binding of virions to the host receptor CD4, but that effect does not differ by tropism. The effect of SLC35A2 on viral fusion, which occurs through engagement with the CCR5 and/or CXCR4 co-receptors, is consistent with the differential infection we previously reported and might contribute to the HIV-1 transmission bottleneck. Novel Strategy to Inhibit HIV by Targeting Integrase/INI1 Interaction Based on the TAR RNA Mimicry Swati Haldar 1 , Updesh Dixit 1 , Emilie Ernst 1 , Rajiv Pathak 1 , Xuhong Wu 1 , Dina Mofed 1 , Vinayaka R. Prasad 1 , David Cowburn 1 , Xiaohe Tong 2 , Asim Debnath 3 , Ganjam V. Kalpana 1 1 Albert Einstein College of Medicine, Bronx, NY, USA, 2 CPC Scientific, Inc, San Jose, CA, USA, 3 New York Blood Center, New York, NY, USA Background: Targeting host-virus interactions is an attractive strategy to overcome the problem of HIV-1 drug resistance. INI1/SMARCB1 is an HIV-1 integrase (IN)-interacting host factor required for assembly and particle production. Studies from our laboratory has indicated that IN binding Rpt1 domain of INI1 (INI1-Rpt1) and viral TAR RNA structurally mimic each other and bind to the same surface on IN ( Dixit et al. Nat. Comm. 2021 ). IN mutants

defective for binding to INI1 are impaired for virion morphogenesis and infection. Because of the INI1-Rpt1 mimicry to TAR, we hypothesize that drugs that target IN-INI1 interaction will be “dual acting”, disrupt both IN-INI1 and IN-TAR interactions, and induce particle morphogenesis defects to inhibit HIV-1 replication. Methods: We used structural information on INI1-Rpt1/IN-(C-terminal domain) CTD complex to generate hydrocarbon stapled peptides derived from interface α1 helix of INI1-Rpt1. These peptides were tested for their effect on IN-INI1, INI-RNA interactions using Alpha Assay and on HIV-1 replication using T-cell lines, PBMC and primary macrophages. The mechanism was tested by assessing their effect on late events and particle morphogenesis, using virology and transmission electron microscopy (TEM) analyses. Results: One of the stapled peptides, SP38, inhibited both IN-INI1 and IN-RNA interactions both in vitro (IC 50 of ~0.3 µM) and in cells. SP38 potently inhibited HIV-1 replication in T-cell line (by 4-5 logs), PBMCs and primary macrophages. SP38 was not cytotoxic and had no effect on expression, proteolytic processing of viral proteins or particle production. However, particles produced in the presence of SP38 were defective for infection, and were impaired for early and late reverse transcription, nuclear localization and integration. TEM analysis indicated that SP38 treatment resulted in morphologically defective “eccentric” virus particles. Furthermore, SP38 inhibited spread of the reactivated virus from latent cells. Conclusions: INI1 α1 helix-derived stapled peptide SP38 dually targets IN-INI1 and IN-RNA interactions and potently inhibits HIV-1 replication by inducing defective particle morphology. These results validate our hypothesis, and provide a proof-of-concept for developing drugs derived from INI1 to simultaneously target IN/INI1 and IN/RNA interactions. SP38 represents a novel first-in-class HIV-1 inhibitor that targets host-virus interactions to inhibit HIV-1 particle morphogenesis. Novel Functions of TRIM21: Post-Translational Regulation of Cytoplasmic Antiretroviral APOBEC3s Aubrey M. Sawyer, Hannah E. Hudson, Isabelle Clerc, Chisu Song, Richard T. D'Aquila Northwestern University, Chicago, IL, USA Background: Apolipoprotein B mRNA editing complex 3 (APOBEC3; A3) enzymes are cytidine deaminases. If packaged into a virion assembling in an HIV-producing cell, A3G, F, and H render that virion non-infectious by hypermutating viral genomes during reverse transcription. HIV’s viral infectivity factor (Vif) recruits these A3s present in the cytoplasm to a Cullin RING ligase (CRL) for ubiquitination and proteasomal degradation, preventing their packaging into virions. Outside of this, little is known regarding A3 post translational regulation. We sought cellular regulators of these A3s as targets for discovery of new agents to limit virion infectivity. Here we identify a novel function of TRIM21 as a post-translational regulator of these anti-HIV A3s. Methods: Bottom-up proteomics was used to identify protein-protein interactors of anti-HIV A3s. Knock down and overexpression of an interactor in T cell lines (CEM, H9) defined effects on A3s. Transfection models were used to characterize the domains of TRIM21 required for activity and if a CRL was needed by using an inhibitor of cullin-activating neddylation (MLN4924) or monoubiquitination (TAK-243). T cell lines with modified interactor expression were infected with HIV to measure effects on replication competent viral production in vitro. Results: A screen revealed tripartite motif containing 21 (TRIM21) as an interactor of the A3s. Co-transfection of A3 proteins with TRIM21 caused degradation of A3G, F, and H. Knockdown of TRIM21 resulted in increased A3G protein levels and overexpression reduced endogenous A3G protein in T cell lines. Treatment of TRIM21-transfected cells with inhibitors of either neddylation or monoubiquitination prevented degradation. Truncation of the TRIM21 c-terminal domains also resulted in loss of A3 degradation. Treatment of T cell lines with interferon and activation of primary T cells via TCR stimulation resulted in increased TRIM21 expression. Vif-positive, replication competent HIV infection of cell lines with TRIM21 knocked down resulted in a 40% reduction in supernatant p24 production compared to the parental cell line. Conclusions: TRIM21 post-translationally regulates cytoplasmic antiretroviral A3s in a manner distinct from its canonical antiviral function. TRIM21 regulation of the A3s is present in physiologically relevant cell models and requires CRL ubiquitination. TRIM21 knockdown reduces HIV production, suggesting future efforts to discover a TRIM21 inhibitor that limits spread of HIV.

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CROI 2025