CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

Methods: We investigated HIV-1-MLO formation in vitro and in vivo during acute infection using an animal model. We used advanced imaging techniques such as double immunogold labeling, electron microscopy, and tomography to analyze viral core structures within HIV-1-MLOs. Functional assays were performed to assess the stability of HIV-1-MLOs under reverse transcriptase (RT) inhibitor treatment and to determine their role in viral genome maturation and immune evasion. Results: Our study demonstrated that HIV-1-MLOs persist for several weeks in infected cells and are essential for canonical post-nuclear entry steps. Within HIV-1-MLOs, we identified distinct categories of viral cores, likely representing different stages of nuclear RT. Importantly, we investigated whether HIV 1-MLOs form in vivo during physiological infection. For the first time, our research using humanized mice revealed that HIV-1 induces biomolecular condensates during acute infection in vivo . HIV-1-MLOs play a critical role in viral genome maturation and immune evasion, either preserving viral infectivity or promoting dormancy in the presence of RT inhibitors by sequestering the viral RNA genome in a stable, inactive state that can later be reactivated. Notably, the restoration of nuclear RT occurs exclusively within these organelles. When HIV-1-MLOs are disassembled, there is a complete loss of viral infectivity, even after the removal of RT inhibitors. During canonical infection, in the absence of drugs, dismantling HIV-1-MLOs exposes newly synthesized vDNA, triggering innate immune responses. Conclusions: HIV-1-MLOs play a critical role in viral genome maturation and immune evasion, maintaining viral infectivity and dormancy in the presence of RT inhibitors by holding the viral RNA genome in a stable, inactive state. These nuclear sites are essential for shielding the newly formed viral dsDNA from host immune responses, ensuring the virus’s ability to persist. Disrupting HIV-1-MLOs presents a potential therapeutic target that could alter latent HIV-1 reservoirs, which are established early during infection. Design of SHIVs Encoding HIV-1 Recombinase Brec1 Recognition Site for Nonhuman Primate Cure Studies Ryan Krause, Edward Kreider, Amie Albertus, Kevin Newcombe, Suvadip Mallick, Weimin Liu, Yingying Li, Emily Lewis, Katharine Bar University of Pennsylvania, Philadelphia, PA, USA Background: The Brec1 recombinase is a genomic engineering approach that shows promise in HIV cure, with in vitro and mouse studies showing efficient excision of HIV provirus. Brec1 recognizes a highly conserved sequence ( loxBTR ) found in the R region of the long terminal repeats (LTRs) that flank the integrated HIV-1 provirus. The loxBTR sequence is found in more than 90% of group M HIV-1, but is not encoded in the SIV strains used in the nonhuman primate (NHP) model of HIV. Here, we design and characterize 3 distinct chimeric SIV/HIV viruses (SHIVs) encoding loxBTR to enable nonhuman primate (NHP) studies of Brec1. Methods: Molecular cloning was used to incorporate loxBTR in three ways into a previously validated SHIV infectious molecular clone (IMC), SHIV.C.CH505.v2: by replacing R (Replace-R), replacing the polyA stemloop within R (Replace PolyA), or inserting loxBTR in U3 (U3-Insertion). loxBTR -IMCs were transfected and assessed for both virion content and infectivity compared to the parental SHIV.C.CH505.v2. Rhesus CD4+ T cells (rhCD4s) were infected and comparative growth kinetics over 15-45 days were captured measuring viral p27. Viral RNA harvested from culture supernatants were sequenced to assess for virus adaptation. Results: All loxBTR -IMCs generated infectious virions. Replace-PolyA and U3-Insertion demonstrated substantial infectious particle production (43% and 52% of parental IU/µL, respectively), and viral fitness (66% - 75% of parental at day 12, respectively), whereas Replace-R showed minimal virion production and replication (2% and 4% at day 12, respectively). Long-term 45-day cultures led to peak p27 levels of 252ng (U3-Insertion), 212ng (Replace-PolyA), and 143ng (Replace-R) at day 42; no viruses demonstrated adaptation within the loxBTR sites. Conclusions: loxBTR -SHIVs produce infectious, replication-competent viruses capable of retaining the Brec1 target site over many rounds of productive infection. To our knowledge, this is the first example of a primate lentivirus that is HIV/SIV chimeric in the essential, polyfunctional regulatory region of R. loxBTR -SHIVs offer a new platform for NHP studies investigating Brec1 recombinase-based HIV-1 cure strategies.

either through knock-out or recruitment of CPSF6 by HIV-1 capsid cores, leads to changes in APA and altered cellular permissivity to HIV-1 infection. Methods: To explore the role of CPSF6 in HIV-1 infection, we leveraged CRISPR Cas9 gene editing to knock out CPSF6 in primary CD4+ T cells. Edited cells were used in spreading infection assays with HIV-1 NL4-3. We assessed the impact of CPSF6 knock-out on gene expression in primary CD4+ T cells using bulk RNA sequencing. We analyzed RNA-sequencing data from CPSF6 knock-out and HIV-1 infected cells using an algorithm to determine changes in poly(A) site usage. Results: CPSF6 knock-out cells were more permissive to HIV-1 NL4-3 infection as compared to non-targeting controls. RNA-sequencing of uninfected CPSF6 knock-out primary CD4+ T cells revealed broad transcriptional rewiring characterized by downregulation of the interferon signaling pathway and changes in expression of known HIV-1 host factors, including TRIM5α. CPSF6 knock-out triggered changes in APA and shortening of 3’ UTR lengths, particularly in genes related to regulation of the innate immune response. We observed that infection of primary CD4+ T cells with HIV-1 strains that bind CPSF6 led to changes in APA and shortening of 3’ UTRs. Furthermore, HT1080 cells, which exhibit a greater degree of relocalization of CPSF6 in cell nuclei in response to HIV-1 infection, undergo more extensive 3' UTR shortening following HIV-1 infection. Conclusions: Together, these data suggest a model in which disruption of CPSF6 function in primary CD4+ T cells leads to transcriptional rewiring driven by changes in APA, resulting in enhanced permissivity to HIV-1 infection. Furthermore, recruitment of CPSF6 by HIV-1 cores or relocalization of CPSF6 during HIV-1 infection may trigger APA and transcriptional rewiring. A Targeted CRISPR Screen Identifies ETS1 as a Regulator of HIV Latency Ashokkumar Manickam 1 , Terry Hafer 2 , Abby Felton 2 , Nancie Archin 1 , David M. Margolis 1 , Michael Emerman 1 , Edward Browne 1 1 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 2 Fred Hutchinson Cancer Center, Seattle, WA, USA Background: Human immunodeficiency virus (HIV) infection is regulated by a wide array of host cell factors that combine to influence viral transcription and latency. Methods: To understand the complex relationship between the host cell and HIV latency, we performed a lentiviral CRISPR screen that targeted a set of host cell genes whose expression or activity correlates with HIV expression. Results: We investigated one of the identified factors - the transcription factor ETS1 and found that it is required for maintenance of HIV latency in a primary CD4 T cell model. Interestingly, ETS1 played divergent roles in actively infected and latently infected CD4 T cells, with knockout of ETS1 leading to reduced HIV expression in actively infected cells, but increased HIV expression in latently infected cells, indicating that ETS1 can play both a positive and negative role in HIV expression. CRISPR/Cas9 knockout of ETS1 in CD4 T cells from ART-suppressed people with HIV (PWH) confirmed that ETS1 maintains transcriptional repression of the clinical HIV reservoir. Transcriptomic profiling of ETS1-depleted cells from PWH identified a set of host cell pathways involved in viral transcription that are controlled by ETS1 in resting CD4 T cells. In particular, we observed that ETS1 knockout increased expression of the long non-coding RNA MALAT1 that has been previously identified as a positive regulator of HIV expression. Furthermore, the impact of ETS1 depletion on HIV expression in latently infected cells was partially dependent on MALAT1. Conclusions: Overall, these data demonstrate that ETS1 is an important regulator of HIV latency and influences expression of several cellular genes, including MALAT1, that could have a direct or indirect impact on HIV expression. HIV-1 Membraneless Organelles Orchestrate Viral Genome Maturation and Immune Evasion Francesca Di Nunzio 1 , Selen M. V. Ay 1 , Julien Burlaud Gaillard 2 , Anastasia Gazi 1 , Philippe Roingeard 2 , Fabrizio Mammano 2 , Yevgeniy Tatirovsky 1 , James Di Santo 1 , Background: HIV-1 establishes unique biomolecular condensates known as HIV-1 membraneless organelles (HIV-1-MLOs) within the nucleus of infected cells. It has been observed that these organelles localize in some nuclear speckles during the early steps of HIV-1 infection. Despite their identification in vitro , questions remain regarding their persistence, composition, function, and formation in vivo . Celine Cuche 1 , Viviana Scoca 1 , Jean-Sebastien Diana 1 1 Institut Pasteur, Paris, France, 2 Université de Tours, Tours, France

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CROI 2025