CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

303

Identification of a New Class of Capsid-Targeting Inhibitors That Specifically Block Nuclear Import Aude Boulay 1 , Emmanuel Quevarec 1 , Isabelle Malet 2 , Giuseppe Nicastro 3 , Valerie Courgnaud 4 , Yves L. Janin 5 , Ian A. Taylor 3 , Anne-Genevieve Marcelin 2 , Laurent Chaloin 1 , Nathalie J Arhel 1 1 Université de Montpellier, Montpellier, France, 2 Hôpitaux Universitaires Pitié Salpêtrière, Paris, France, 3 The Francis Crick Institute, London, United Kingdom, 4 Institut de Génétique Moléculaire de Montpellier, Montpellier, France, 5 Centre National de la Recherche Scientifique, Paris, France Background: Nuclear localized viral genomes account for the persistence HIV-1 in patients despite successful antiretroviral therapy. Drugs that target reverse transcription or integration do not prevent HIV from reaching the nucleus and producing viral transcripts and proteins. Inhibiting HIV-1 nuclear import could make a significant contribution by reducing the reservoir. Methods: A total of 85 compounds and structural analogues were tested following an in silico screen of compounds that recognize the previously described TRN-1 binding pocket on HIV-1 capsid (Fernandez et al. Nat Microbiol 2019). Antiviral activity was assessed in cell lines and primary human lymphocytes, using X4- and R5-tropic molecular clones, clinical isolates from treatment-naive patients, and ART-resistant mutants. Toxicity and pharmacokinetic properties were obtained in primary human lymphocytes and humanized mice. Co- immunoprecipitation, surface plasmon resonance, proximity ligation assays, and methyl-Trosy NMR, were used to quantify binding to CA and TRN-1; quantitative PCR to analyse reverse transcripts, 2-LTR circles and integrated provirus; and confocal imaging to measure capsid nuclear import. The effects on uncoating were tested in vitro and in cells, and a total of 16 capsid variants from divergent lentiviruses or stability mutants were compared. Control drugs were NVP, RAL, 3TC, DRV, DTG, PF-74 and LEN. Results: Six compounds (hit H27 and 5 out of 45 structural analogues) inhibited HIV-1 in a dose-dependent manner. The IC 50 was in the low micromolar range and CC50 values were >100μM, indicating a high selectivity index. All 6 compounds inhibited the nuclear import of HIV-1 specifically, without impacting other steps of the viral life cycle such as reverse transcription or particle production. Although H27 reduced the TRN-1-CA association, binding sites were distributed over the entirety of CA. All tested HIV-1 molecular clones, clinical isolates, stability or PF74/LEN-resistant mutants, and resistant mutants to NRTI, NNRTI, PI and INSTI, were sensitive to H27 (25 viruses in total). In contrast, HIV-2, SIVagm and SIVmac were resistant, and infection with the hypostable P38A mutant was even stimulated by H27. Escape mutations to H27 first appeared at day 49 in cell culture and were stability mutants E45L/G46A. Conclusion: This study identifies a new family of capsid-targeting compounds that specifically inhibit HIV-1 nuclear import by modulating the stability of capsid and its ability to bind to nuclear import factors. Impact of Capsid Polymorphisms on Viral Fitness and the Susceptibility to Lenacapavir Derek Hansen , Silvia Chang, Stephen Yant, Ross Martin, Thomas Aeschbacher, Arthur Cai, Jason Perry Gilead Sciences, Inc, Foster City, CA, USA Background: Lenacapavir (LEN) is a first-in-class, long-acting capsid (CA) inhibitor for the treatment and prevention of HIV-1 infection. While LEN has shown full potency against different HIV-1 subtypes, it remains unclear how viral diversity encountered in the clinic may impact its efficacy. Herein we analyzed HIV-1 CA sequence diversity to identify natural polymorphisms within the LEN binding site and assessed each for their impact on viral fitness and susceptibility to LEN. Methods: CA binding site residues within a given radius of LEN were identified in Pymol. HIV-1 CA sequences from public (N=9232) and Gilead trial datasets (N=825) were analyzed for naturally occurring binding site polymorphisms across subtypes A1, B, C, D, F1, G, CRF01_AE and CRF02_AG. Site-directed mutants encompassing CA polymorphisms with a > 0.5% prevalence were expressed as single-cycle NL4.3- based reporter viruses. Infectivity and antiviral EC 50 values for WT and mutant viruses were determined in MT- 4 cells. Results: Of the 25 CA amino acids identified within a 5Å radius of LEN at its binding site, 10 (40%) were completely invariant among the >10K unique HIV-1 CA sequences analyzed. Half (5/10) of these conserved residues (M66, Q67, K70, N74 and A105) matched those previously associated with LEN resistance when mutated. Among each of the remaining 15 LEN binding site residues, at least 1 variant was identified across the 8 subtypes evaluated, with codons S41, Q50, T54 and N183 being the most variable with 6, 8, 7 and 9 substitutions detected,

respectively. Site-directed HIV-1 reporter viruses encompassing all of the observed CA variants (n=48) were produced and evaluated for infectivity and drug susceptibility in MT-4 cells. Approximately half (25/48) of these mutants showed impaired infectivity (<50%, range 0.006 – 47%) relative to the WT, with 6 (I37Y, Q50P, N53K, T54Y, I73F, R173K) being so severely impaired that it prevented LEN resistance profiling. Of the remaining 42 CA variants, 39 (93%) remained fully susceptible to LEN (FC=0.6- 2.4). Three variants (Q50E, L56V and N57H), with prevalence of ~0.5% in a single subtype (C or D) and impaired infectivity (0.6-25% of WT), showed reduced susceptibility to LEN relative to WT (FC=3.1, 72 and 4890, respectively). Conclusion: With few exceptions, our mutant HIV panel comprising rare naturally occurring LEN binding site variants in CA remained fully susceptible to LEN, suggesting that the existing natural viral diversity should minimally impact LEN efficacy in the clinic. Massive Endocytosis Mechanisms Are Involved in CD169-Mediated Uptake of HIV-1 by Dendritic Cells Fernando Laguía-Nueda 1 , Jakub Chojnacki 1 , Itziar Erkizia 1 , Elena Rebollo 2 , Carlos Enrich 3 , Maria Isabel Geli 2 , Javier Martinez-Picado 1 , Patricia Resa-Infante 1 1 IrsiCaixa Institute for AIDS Research, Badalona, Spain, 2 IBMB – Molecular Biology Institute of Barcelona, Barcelona, Spain, 3 Universitat de Barcelona, Barcelona, Spain Background: Myeloid cells, such as monocytes, macrophages, and dendritic cells (DCs), are main sentinels of the immune system against invading viruses. However, HIV-1 can take advantage of this to disseminate throughout the body. This process relies on the recognition of gangliosides on the viral envelope by the CD169/Siglec-1 receptor expressed on the cell surface of activated myeloid cells. This interaction triggers the internalization of HIV-1 in a structure named Viral Containing Compartment (VCC), where viral particles remain infectious. Although VCC formation process is largely unknown, previous data suggest that it is independent of clathrin and requires cholesterol in the cell membrane, as well as transient deregulation of cortical actin. Therefore, we hypothesized that HIV-1 exploits the machinery used during Massive Endocytosis (MEND) to enter dendritic cells after binding to CD169. Methods: Live cell confocal imaging of DCs was conducted to obtain a 3D reconstruction of VCC formation and measure its dimensions and dynamics. Actin and cholesterol staining was performed in fixed and live cells to assess their roles during this process. PI3K inhibitors and protein palmytoilation inhibitors were used to evaluate their effect in the uptake of Viral Like Particles (VLP) by confocal microscopy. Images were processed with ImageJ and data analysis with GraphPad. Results: HIV-1 VLP uptake dynamics and VCC dimensions do not match with classical endocytic pathways, but VCC size (2.9 ± 0.7 μm diameter and 20 ± 9.9 μm3 volume) indicates massive plasma membrane invagination. Although actin was not observed by phalloidin staining in the early steps of VCC formation, it is essential for VCC maintenance. PI3K and protein palmitoylation inhibition arrested the process before VLP entry, reducing the rate of VCC formation in DCs from 70% to 40% and 20%, respectively. Strikingly, cholesterol coalescence determined VCC formation as the cholesterol probe signal was doubled in the VLP uptake regions, as observed by live cell imaging. Conclusion: These data suggest that MEND mechanisms participate in the internalization of viral particles and subsequent formation of VCC in DCs. This work provides new insights into the interaction of HIV-1 with myeloid cells revealing new therapeutical targets to hinder virus dissemination. Blocking VCC formation offers potential cross-protection against enveloped viral infections that use CD169 receptor, such as Ebolaviruses and other hemorrhagic fever viruses. Antagonism of Viral Glycoproteins by Guanylate-Binding Protein 5 Hana Veler , Geraldine V. Vilmen, Abdul A.Waheed, Cheng Man Lun, Eric O. Freed National Cancer Institute, Frederick, MD, USA Background: Guanylate binding protein (GBP) 5 is an interferon-inducible cellular factor with broad antiviral activity, reducing the infectivity of progeny virions by interfering with processing and incorporation of viral glycoproteins. GBP5 is believed to inhibit the infectivity of HIV-1, highly pathogenic influenza A, and dengue virus by reducing the proteolytic activity of furin. However, the exact mechanism by which GBP5 inhibits processing of viral glycoproteins and whether it only affects furin-dependent glycoproteins remains poorly understood.

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CROI 2024