CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

out by studying ASP nuclear import and its impact on DNA repair, as well as the impact of ASP on mitochondrial function. We used computational methods to identify the evolutionary mechanisms that created the asp gene and its association with HIV-1 disease progression. Results: We created asp deficient HIV-1 strains by mutating the start codon or by introducing early stops via single nucleotide substitutions that lead to synonymous codons in env. ASP-deficient strains show reduced fusion and replication capacity compared to wildtype. To determine potential mechanisms, we identified ASP binding partners by MS and validated the results by co-IP. We found that the Transportin 1 pathway mediates ASP nuclear import, and that in the nucleus ASP interacts with factors involved in multiple DNA damage repair pathways. Functional studies confirmed that ASP promotes DNA repair. Also, ASP interacts with and increases the expression of mitochondrial factors of the oxidative phosphorylation and ATP biosynthesis pathway. Finally, sequence analyses show that creation of asp occurred via evolution of codon usage in env involving differential use of synonymous codons or conservative amino acid substitutions that eliminated early stops in asp. While presence of asp constrains env genetic diversity, it provides a selective advantage as the presence of asp is significantly more frequent in rapid progressors than long term nonprogressors. Conclusion: For the first time our studies link a full-length asp ORF to HIV-1 entry and replication in vitro and disease progression in vivo. We identified possible molecular mechanisms including impact of ASP on DNA repair and mitochondrial function, and the evolutionary mechanisms that created asp. In Vivo Detection of HIV-1 Antisense Transcripts in Donors Before and During ART Adam A Capoferri 1 , Toluleke O. Famuyiwa 1 , Rachel Sklutuis 1 , Sachi Pathak 1 , Jennifer L. Groebner 1 , Rui Li 2 , Jason W. Rausch 1 , Steven G. Deeks 3 , John W. Mellors 4 , John M. Coffin 5 , Fabio Romerio 2 , Mary F. Kearney 1 1 National Cancer Institute, Frederick, MD, USA, 2 The Johns Hopkins University School of Medicine, Baltimore, MD, USA, 3 University of California San Francisco, San Francisco, CA, USA, 4 University of Pittsburgh, Pittsburgh, PA, USA, 5 Tufts University, Boston, MA, USA Background: Natural antisense transcripts (NATs) are expressed by viruses, prokaryotes, and eukaryotes and primarily function in regulating sense gene expression through multiple mechanisms. In vivo studies have shown that HIV-1 expresses antisense transcripts (AST) from a Tat-independent negative sense promoter in the 3' LTR. In cell line studies, AST was demonstrated to promote HIV latency through epigenetic modification of histones in the 5' LTR by the Polycomb Repressor Complex 2 (PRC2). Here, we asked whether HIV AST is expressed in infected PBMCs collected from untreated and ART-treated donors. Methods: PBMCs were obtained from 10 donors in the SCOPE cohort and 2 donors from University of Pittsburgh. Untreated donors were either ART-naïve or undergoing treatment interruption with viral loads ranging from <50– 200,000 copies/ml of plasma (n=10). Donors on ART had viremia suppressed (<50 cp/ml) for a median of 5.4-years (n=3). AST levels were measured by cell associated antisense RNA single-genome sequencing (SGS) of a 1.7kb fragment in the opposite orientation of the env coding region. An endpoint digital PCR approach with tagged-cDNA and donor-specific primers was also used to quantify AST copies in the samples. Results: We detected HIV AST in 11/12 donors with a median of 14 [IQR 5-34] copies/100 infected PBMCs (Figure-Left Panel). Antisense SGS revealed that about 5% of infected PBMCs collected from donors on ART contained AST at any given time. The genetic diversity of the antisense transcripts was consistent with expression from a diverse population of proviruses. Proviral clonal populations were identified with matched HIV AST from multiple aliquots of single-infected PBMC (Figure-Right Panel). Digital PCR showed similar levels of AST expression in untreated donors with varying levels of plasma viremia. Further, in the donors on ART, we observed no statistical difference between the levels of sense and antisense transcripts. Conclusion: As in the case of other NATs, HIV antisense transcripts are expressed at low levels in both ART- treated and untreated individuals. The in vivo expression of AST irrespective of treatment status warrants further investigation into its potential role as a long non-coding RNA capable of regulating HIV-1 sense gene expression and inducing HIV latency. Understanding the role of HIV AST in vivo may inform future strategies for controlling HIV replication without ART.

previous report, we found that A3G mRNA splicing defect is independent of the insertion of an Alu element in the A3G locus. Instead, it is strongly associated with a small intronic variation in only rhesus macaques and other similar nonhuman primates used frequently in HIV studies. Conclusion: Taken together, our studies have unveiled and characterized a significant difference in A3G-mediated anti-HIV/SIV immunity across human and nonhuman primate models. These findings have significant implications for our understanding of the disparities in viral restriction, drug resistance and immune evasion mechanisms between humans and nonhuman primate models of HIV. High Replication Fitness and Potent Innate Immune Evasion Function of a HIV-1 VB Strain Dorota J Kmiec , Kerstin Regensburger, Frank Kirchhoff Ulm University Medical Center, Ulm, Germany Background: A highly virulent subtype B (VB) HIV-1 has been detected in >100 individuals (Wyant et al., 2022). On average, infected patients show 5-fold higher viral loads and 2-fold faster CD4+ cell loss compared to other subtype-B infected individuals. To determine molecular determinants of its virulence, we functionally characterized a clone (VB_6) of this HIV-1 variant. Methods: HIV-1 VB_6 isolate sequence was found to be most similar to VB consensus and used to synthesize an infectious molecular clone. Following transfection into HEK293T cells to generate infectious virus, particle infectivity, cold stability and ability to infect primary T cells and monocyte-derived macrophages were examined. Replication kinetics of VB_6 in activated T cells were compared to 8 other HIV-1 subtype B strains. Sensitivity to CCR5 and CXCR4 inhibitors was measured in primary T cells by flow cytometry. Resistance to IFI16 and activation of NF-кB reporter were tested by co-transfection of the molecular clone into HEK293T cells. Induction of cell death, modulation of immune receptors and proinflammatory cytokine release by VB_6 were determined by flow cytometry. Results: VB_6 had average particle infectivity and stability, and like most primary subtype B strains is T-cell tropic and utilized CCR5 entry co-receptor. However, the replication fitness of VB_6 in T cells was superior to all tested (8) primary subtype B CCR5-tropic strains, including 3 transmitted-founder viruses. VB_6 potently downmodulated immune receptors (CD4, CCR5, CXCR4 and tetherin) from the cell surface using Vpu and Nef in a more efficient manner than the NL4-3 reference strain. VB_6 infection of primary T lymphocytes induced increased levels of cell death and proinflammatory cytokines, while suppressing type I and III IFN production. In addition, VB_6 was more active at inducing NF-kB signaling and more resistant to IFI16 than most other tested subtype B strains. Conclusion: The hyper-virulent HIV-1 VB_6 strain shows high replicative fitness in primary human T lymphocytes and expresses Vpu and Nef proteins that potently downmodulate immune receptors. VB_6 is also adept at downmodulating tetherin and shows reduced sensitivity to inhibition by IFI16. In line with increased replication, VB_6 infected primary T cells produce more proinflammatory cytokines and show reduced viability. A combination of potent immune evasion and high replicative fitness likely contribute to the virulence of VB HIV-1. The HIV-1 Protein ASP Promotes Viral Replication and Is Associated With Faster Disease Progression Mohd Shameel Iqbal 1 , Myriam Houmey 2 , Ziyan Xu 1 , Yuchu Wang 1 , Kabir Khan 1 , Isabella Caico 1 , Rui Li 1 , Jean-Michel Mesnard 2 , Nathalie Chazal 2 , Angelo Pavesi 3 , Fabio Romerio 1 1 The Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2 Université de Montpellier, Montpellier, France, 3 University of Padova, Padova, Italy Background: Decades after its discovery, the HIV-1 antisense gene asp remains an enigma. Asp overlaps env and is present exclusively in pandemic group-M HIV-1 strains. Thus, its creation may have improved HIV-1 fitness or spread. We reported that ASP is found on the surface of HIV-1 virions. We studied how ASP expression impacts HIV-1 entry and replication and the underlying mechanisms. We also studied the evolutionary mechanisms that created asp, and its association with disease progression. Methods: We used fusion and infection assays to assess how ASP expression impacts HIV-1 entry and replication. Identification and molecular validation of ASP interacting partners were performed by mass spectrometry (MS) and co-immunoprecipitation (Co-IP). Functional validation of MS results was carried

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CROI 2024