CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

POSTER ABSTRACTS

300

Targeting Tat-Dependent Transcriptional Rewiring to Manipulate HIV Latency William Cisneros, Shimaa Soliman, Miriam Walter, Eun-Young Kim, Ali Shilatifard, Steven M. Wolinsky, Judd F Hultquist Northwestern University, Chicago, IL, USA Background: Integrated HIV-1 proviruses rely on host transcriptional machinery for RNA expression. To enhance transcriptional activity, the virus encodes the transactivating protein Tat, which recruits positive transcription elongation factor b (P-TEFb) to sites of nascent proviral transcription through recognition of the TAR RNA stem loop. P-TEFb phosphorylates the C-terminal tail of RNA polymerase II (RNA Pol II) to enhance processivity and trigger transcriptional elongation. During normal cellular transcription, P-TEFb is recruited to RNA Pol II by the joint action of the PAF1 complex (PAF1C) and the Super Elongation Complex (SEC). Given the apparent functional redundancy between these complexes and Tat, we set out to determine their role in HIV-1 replication and latency. Methods: HIV-1 spreading infection was monitored following genetic or chemical perturbation of these complexes in primary CD4+ T cells. For genetic perturbation, CRISPR-Cas9 gene editing was used to knock-out each complex member in cells from independent donors. For chemical perturbation, we developed and validated first-in-class small molecule inhibitors of the SEC and PAF1C, which were delivered prior to challenge. These small molecule inhibitors were also tested for their impact on latent proviruses in cell line models of latency and in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH). Results: Both genetic and chemical perturbation of the SEC and PAF1C significantly increased HIV-1 replication in primary CD4+ T cells, suggesting that these complexes inhibit viral replication. In J-Lat models of latency, neither inhibitor was sufficient for latency reactivation, though they did act synergistically with other latency reversing agents (LRAs). In contrast, both inhibitors significantly increased the expression of HIV-1 gag in PBMCs from PLWH, both individually and with other LRAs. Mechanistic studies suggest that the latency reversing activity of these compounds is Tat-dependent, and that they otherwise promote latency due to an inability to recruit P-TEFb. Conclusion: These results demonstrate that the SEC and PAF1C inhibit HIV-1 replication and serve as blocks to Tat-dependent transcription in latently infected cells. Small molecule inhibitors of these complexes reactivate latent proviruses in patient PBMCs and act synergistically with other known LRAs. Future directions will explore the Tat-dependency of these compounds and their potential as dual-acting latency reversing and promoting agents.. Impact of HIV-1 TAR Sequence Diversity on Its RNA Secondary Structure in HIV-1 DNA Genomes Mohith Reddy Arikatla 1 , Pragya Khadka 1 , Zheng Tang 1 , Erika Benko 2 , Colin Kovacs 2 , Marina Caskey 3 , Taddeo Kityamuweesi 4 , Paul Bbuule 4 , Stephen Tomusange 4 , Aggrey Anok 4 , Jeffrey Martin 5 , Melissa Smith 6 , Timothy J. Wilkin 1 , R. Brad Jones 1 , Guinevere Q. Lee 1 1 Weill Cornell Medicine, New York, NY, USA, 2 Maple Leaf Medical Clinic, Toronto, Canada, 3 The Rockefeller University, New York, NY, USA, 4 Rakai Health Sciences Program, Kalisizo, Uganda, 5 University of California San Francisco, San Francisco, CA, USA, 6 University of Louisville, Louisville, KY, USA Background: HIV-1 TAR is an RNA secondary structure located at the 5' end of the viral transcript. It binds to HIV-1 Tat protein to facilitate viral transcript elongation and plays a role in virologic rebound during therapy cessation. Here, we hypothesize that TAR secondary structures and stability significantly differ across viral subtypes and across intact versus defective HIV-1 DNA genomes. Methods: Near-full-length HIV-1 DNA genome sequences from 47 study participants from Uganda (n=28, subtype A1, C, D), Canada and the US (n=19, subtype AE, B) were obtained using nested PCR (HXB2 638- 9632) and Illumina sequencing; TAR was inferred from the 3'LTR. Each genome was classified as intact or defective (e.g., containing hypermutations and/or large deletions) using the software HIVSeqinR. To obtain TAR associated with transcription

competent genomes, cell-associated viral mRNA transcripts were sequenced from six matching participants (subtype B n=5, AE n=1) using PacBio IsoSeq. TAR secondary structure and its stability as measured by Gibbs free energy (ΔG) values were inferred using Quikfold; a higher ΔG is associated with decreased TAR stability. Results: In the HIV-1 DNA genome analysis, among the 47 study donors (1970 TAR sequences), TAR nucleotide diversity associated with intact viral genomes varied up to 13% interhost and 3% intrahost. Subtype D intact genomes had the least stable TAR relative to A1 and B (p<0.004). TAR in subtype A1 and AE did not share the same secondary structure as subtype B, C and D (linear versus bent hairpins). Per donor, ΔG of TAR associated with intact genomes did not differ from any defective genome categories except was higher when associated with hypermutated genomes (p<0.0006). In the ex vivo viral mRNA transcript analysis, we detected hypermutated genomes that were actively producing viral transcripts which were associated with significantly higher ΔG than non-hypermutated transcripts (p<0.0001), whereas TAR associated with non hypermutated transcripts had similar ΔG values relative to TARs in the intact viral DNA pool from the same donor (p=0.5). Conclusion: Our study reveals HIV-1 TAR genotypes are not identical across viral subtypes or between genome-intact versus defective viruses, and they can be genetically diverse intrahost. Differences in TAR stability may imply differences in the extent of viral transcription activity. Future studies should examine whether TAR stability can predict the quantity of viral transcripts produced. Crosstalk Between Resistance to the HIV-1 Capsid Inhibitor Lenacapavir and Viral Fitness Binh Nguyen , Alex Kleinpeter, Eric O. Freed National Cancer Institute, Frederick, MD, USA Background: Lenacapavir (LEN) is the first capsid inhibitor to be FDA-approved for HIV-1 treatment. Despite high potency and slow-release kinetics, a significant drawback of LEN is its low barrier to viral resistance. A mutation in the HIV-1 capsid, M66I, confers > 80,000-fold resistance to LEN and has been observed in cultured cells and in HIV-1-infected individuals treated with LEN. However, in the absence of LEN, M66I causes a substantial defect in viral fitness (<5% infectivity relative to WT). Given the high mutation rate of HIV-1, it is important to understand how HIV can adapt to circumvent the M66I-induced fitness defect before compensatory mutations are manifested in patients. Methods: We propagated LEN-resistant HIV-1 mutants (pNL4-3) in T-cell lines (SupT1 and MT4) to select for compensatory mutations. Replicating viruses were sequenced to identify compensatory mutations. Identified mutations were sub-cloned into pNL4-3 to examine their effects on virus infectivity and drug sensitivity. Results: M66I propagation in T-cells repeatedly led to WT reversion (I66M). We examined the effects of mutating M66 to other amino acids and determined whether these substitutions recapitulate the behavior of M66I, specifically its resistance to LEN and fitness defect. Of the M66 mutants examined, M66L, M66V, and M66F exhibited similar infectivity defects evident in M66I, but only M66V displayed high-level resistance to LEN. Propagation of these M66 mutants led to several second-site mutations. Of note, H12Y, in combination with A105T and other capsid substitutions, resulted in a >10-fold rescue of M66L infectivity. Conclusion: This study investigates viral escape strategies of M66I, a highly LEN-resistant but fitness- impaired HIV-1 mutant. As a clinically significant variant, this work will reveal important insights into how HIV-1 may maintain LEN resistance while bypassing the fitness defect inherent to M66I.

Poster Abstracts

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CROI 2024