CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

319

Convergence of NF-κB Pathways Increases HIV-1C Transcriptional Fitness Hrimkar B. Buch, Aboli Varunjikar, Udaykumar Ranga Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India Background: HIV-1 subtype C (HIV-1C) has a unique genetic characteristic of incorporating additional NF-kB binding motifs within its long terminal repeat (LTR) region, which enhances its transcriptional strength and replication fitness. Unlike other HIV-1 subtypes, HIV-1C demonstrates a subtype-specific interaction between its central NF-kB and Sp1III motifs. Our recent research identified nine emerging HIV-1C LTR variants from India, exhibiting co-duplication of NF-kB motifs along with upstream LEF and RBEIII sites, known to suppress transcription. This co-evolution of transcription-enhancing (NF-kB) and suppressive (LEF, RBEIII) motifs is novel and significant for understanding viral latency and transcription regulation. We hypothesize that the presence of both classical H-kB and unique C-kB motifs in HIV-1C LTR provides an advantage by optimizing receptivity to classical and alternative NF-kB signals. This adaptability is specific to HIV-1C, distinguishing it from other subtypes. Our study explores whether NF-kB motif polymorphisms in HIV-1C LTR allow efficient integration of these pathways and enhance latency reversal compared to other subtypes. Methods: Jurkat cells were treated with AZD5582 to selectively activate the alternative NF-kB pathway. Activation was confirmed by p100 processing into p52, while IκBα levels confirmed no classical pathway activation. Jurkat cells infected with HIV-1B or HIV-1C LTR reporter viruses were treated with AZD5582 (Figure1-A). Additionally, primary CD4+ T cells infected with a dual-reporter HIV-1 virus were treated with AZD5582, TNF-α, or PMA. Results: Flow cytometry showed a higher response in HIV-1C LTR to alternative NF-kB signaling than HIV-1B (Figure-1B). Immunoblotting confirmed p52 processing in CD4+ T cells treated with AZD5582, with increased reactivated cells observed via flow cytometry, indicating enhanced transcription and latency reversal potential in HIV-1C (Figure-1C). Conclusions: In conclusion, our results suggest that HIV-1C LTR integrates both classical and alternative NF-kB signals more effectively than HIV-1B, contributing to its transcriptional superiority and replication fitness. Future research will assess clinical samples to further compare HIV-1B and HIV-1C LTR responses, potentially informing novel strategies for HIV-1 latency reversal and cure. Fatty Acid Synthesis Controls Both Cellular Susceptibility to HIV and New Particle Production Joshua A. Acklin, Erika Pontillo, Ming Ye, Bruce Walker, Alison Ringel Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Background: As an obligate intracellular pathogen, HIV must hijack resources from infected host cells to support the viral lifecycle. While this includes a viral dependency on host metabolism, little is known about the specific metabolic networks that are essential for infection and viral replication. Previous work leveraging a single-cycle reporter virus has established that blocking the movement of carbon into the TCA cycle from either glucose or glutamine reduces HIV infectivity. In addition to its bioenergetic role, the TCA cycle also serves as a major biosynthetic hub for the synthesis of critical metabolites, like fatty acids. Here, we test the hypothesis that the synthesis of fatty acids is a metabolic dependency during HIV infection. Methods: To identify the role of fatty acid synthesis during HIV infection, we conducted the following analyses: 1) We tested if the expression of the enzymes involved in synthesizing fatty acids is altered during infection using flow cytometry; 2) We tested the impact of blocking fatty acid synthesis on infection in primary T cells through in vitro perturbational studies; and 3) We have begun to mechanistically map the impact of inhibiting fatty acid synthesis on infection by studying the end product palmitate through metabolite tracing and other biochemical approaches. All experiments were conducted at minimum in technical triplicate and with three experimental replicates where possible. Results: Through these studies, we identified that the expression of fatty acid synthetic enzymatic machinery is significantly increased during HIV infection of human T cells. Surprisingly, chemical inhibition of fatty acid synthesis increased the susceptibility to infection (3-to-4 fold). However, analysis of viral particle production by p24 ELISA revealed that particle production on a per-cell basis was significantly decreased. Mechanistic studies have demonstrated that this phenotype is dependent on palmitate, the product of fatty acid synthesis. The figure, table, or graphic for this abstract has been removed.

Conclusions: Our studies indicate that the ability to synthesize fatty acids is a critical metabolic process that can control the balance between infectivity and new viral particle production. While we have identified a role for palmitate in this process, future work will aim to determine the precise biochemical mechanism for this alteration in susceptibility, as well as how in vivo alterations to fatty acid synthesis may affect the clinical progression of HIV infections. Direct Sequencing of Full-Length HIV-1 RNA Reveals the Functional Significance of Site-Specific m6As Ga-Eun Lee, Sarah Golconda , Sanggu Kim The Ohio State University, Columbus, OH, USA Background: While the significance of chemical modifications on RNA has been well established, their specific roles in HIV-1 replication remain unclear and, at times, controversial. Previous studies have provided only low-resolution, type-specific RNA modification site mapping and have relied on indirect analyses of phenotypic effects through the overexpression or knockdown (knockout) of host effectors. These approaches fail to account for the site specific and context-dependent functions of viral RNA modifications. Methods: We conducted full-length, nucleotide-resolution mapping and characterization of chemical modifications on HIV-1 RNA using novel Nanopore direct RNA sequencing (DRS) and oligonucleotide LC-MS/MS methods, which we recently developed (Baek et al., Nature Microbiology , 2024; Baek et al., Methods & Protocols , 2024). Site-directed mutagenesis was employed to characterize the functional properties of HIV-1-specific chemical modifications. Results: Our data revealed a surprisingly simple HIV-1 modification landscape, with three dominant site-specific N6-methyladenosine (m6A) modifications near the 3’ end, out of the 242 known m6A motifs (DRACH) on the HIV-1 RNA genome. Each of these three m6A marks up to 89% of HIV-1 RNAs, a level of stoichiometry exceptionally higher than that seen in typical cellular mRNAs (typically less than 20%). Knocking out one of these three m6As did not affect HIV-1 RNA production, isoform expression, viral protein expression, virion production, or infectivity. However, knocking out all three m6As (triple mutation) resulted in a significant reduction in full-length unspliced HIV-1 RNA and Gag expression, while increasing the fraction of completely spliced RNAs. Despite this apparent “over-splicing” phenotype, our single-RNA-level analysis revealed that the more m6As deposited in individual HIV-1 RNAs, the more splicing events occurred in those RNAs. These counterintuitive results can be explained by the additional role of these m6As in regulating the expression of HIV-1’s Rev protein, which is crucial for controlling unspliced RNA and Gag expression. Conclusions: The potential HIV-1-specific and context-dependent roles of m6As have been overlooked in previous studies. Our data shed new light on the functional and evolutionary significance of m6As in HIV-1 biology. The technological innovations presented here will also serve as valuable tools for future exploration of the complex RNA biology of HIV-1. Functional Study of N6-Methyladenosine (m6A) Machinery in HIV Infection of Primary CD4+ T Cells Lacy M. Simons 1 , Kathryn A. Jackson-Jones 1 , Abolfazl Arab 2 , Albertas Navickas 2 , Aubrey M. Sawyer 1 , Hani Goodarzi 2 , Judd F. Hultquist 1 1 Northwestern University, Chicago, IL, USA, 2 University of California San Francisco, San Francisco, CA, USA Background: N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional modification of cellular and viral RNA and is critical to the regulation of its localization, stability, and translation. The core m6A machinery consists of writers that deposit the modification, erasers that remove it and readers that respond to the presence of m6A on RNA. Previous studies on the role of m6A during Human Immunodeficiency Virus (HIV) infection have produced conflicting results, particularly in regard to which readers influence HIV replication. Due to the significant functional variation of m6A modifications between cell types, we aimed to characterize the role of m6A during HIV infection using primary CD4+ T cells. Methods: The expression of the core m6A machinery in primary CD4+ T cells was assessed using qPCR assays. Systematic knockout of 46 genes implicated in m6a modification was carried out using a CRISPR-Cas9 system in primary CD4+ T cells. Edited cells were subjected to a spreading infection using a HIV-1 NL4-3 GFP reporter virus; and subsequently screened by flow cytometry to quantify infection levels. We then carried out m6A-methylated RNA

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CROI 2025