CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

Results: Within the 896 proviral genomes sequenced, 539 unique deletion sites were identified. Deletion length ranged from 2-8884 bases, with 76% of deletions >4000 bases in length. Deletion start and stop sites were non- randomly distributed along the HIV genome. Most deletion start sites occurred at the 5’ end of the provirus (76% between positions 666-4000, 5’ U5 LTR to pol). Most deletion stop sites occurred at the 3’ end (80% between positions 8000-9676, env to 3’ U5 LTR). Additionally, 9.3% of deletion start sites occurred between positions 4750-4950, a region that encompasses the central polypurine tract (cPPT). Investigation of nucleotide context found a significant association (p<0.001) between short sequence repeats and deletion start and stop sites, indicating that nucleotide homology at deletion sites is not due to chance. Conclusion: This study showed that deletions in HIV proviruses occur at non-random sites, indicating they are generated by a specific mechanism. The presence of short sequence repeats at deletion junctions (an important factor for the generation of recombinants) and the identification of a common deletion site at the cPPT (a known recombinant site) suggests deletions occur as a result of RT-mediated template switching. Understanding the mechanisms that generate defective proviruses will be important for developing future eradication methods that enhance their production. 170 HOST FACTORS INFLUENCE SIV AND HIV-1 INFECTION AND SENSITIVITY TO CAPSID INHIBITORS Rachel Scheck, Augustin P. Twizerimana, Zeli Zhang, Dieter Häussinger, Carsten Münk Heinrich Heine University Hospital, Düsseldorf, Germany Background: SIVs of chimpanzees (cpz) and gorillas (gor) rarely established infections in humans in which these viruses were further transmitted from human to human. The viral capsid (CA) is the key viral determinant of primate lentiviruses that are targeted by cytoplasmic proteins such as cyclophilin A, TRIM5α, CPSF6 (mRNA processing protein cleavage and polyadenylation specificity factor 6) and MX2 that affect infection and likely regulate cross- species transmission. Methods: In order to characterize the impact of different cell types and dependency pathways versus restriction pathways, luciferase reporter viruses for SIVcpz (for both SIVcpzPtt and SIVcpzPts), SIVgor and rare HIV-1 N, O and P were constructed. Infection experiments using VSV-G pseudotypes were performed in the presence and absence of host proteins and pharmacological inhibitors (e.g. cyclosporine A, PF74). Results: Here we show that small inhibitors of the viral capsid (PF37, PF74) differently effect the infection of SIVcpz and HIV-1s in human and non-human cells. While SIVcpzPtt was sensitive to PF37 inhibition in human HOS and HeLa cells, SIVcpzPts were only inhibited in HOS cells. No SIVcpz were blocked in rhesus monkey cells by PF37. In contrast, HIV-1 M was sensitive to PF37 in all three cell types. We constructed a SIVcpzPtt with the capsid of the Pts virus. The chimeric SIVcpz lost only partially the sensitivity to the capsid inhibitor. Conclusion: Manipulation of the viral infection by inhibitors for capsid is strikingly dependent on the cell-type. PF37 and related capsid inhibitors can inhibit non-HIV-1 primate lentiviruses. This inhibition, however, requires unidentified cellular host factors that differentially interact with HIV-1 M, and SIVcpz. PF37/PF74-sensitivity of SIVcpz is only partially regulated by the viral capsid. 171 VPX INDUCES AN IFN-RELATED INNATE IMMUNE RESPONSE DISTINCT FROM SAMHD1 ABROGATION Maria Pujantell 1 , Eva Riveira-Muñoz 1 , Alba Ruiz 1 , Roger Badia 2 , Bonaventura Clotet 1 , Jose A. Este 1 , Ester Ballana 1 1 IrsiCaixa Institute for AIDS Research, Badalona, Spain, 2 Institute for Health Science Research Germans Trias i Pujol, Badalona, Spain Background: SAMHD1 is an HIV restriction factor that acts by depleting the intracellular pool of nucleotides, a process that is counteracted by the virion- packaged accessory protein Vpx, through SAMHD1 proteosomal degradation. SAMHD1 mutations lead to Aircardi-Goutieres syndrome characterized by increased IFN production. SAMHD1 depletion has also been associated to aberrant DNA production and production and innate immune activation. Here, we investigate the interplay between SAMHD1 depletion, innate immune activation and susceptibility to HIV-1 infection. Methods: CD14+ human monocytes were differentiated to macrophages. Knockdown of SAMHD1 was achieved by RNA interference or by transducing macrophages with VLP-containing HIV-2 Vpx. A SAMHD1 knockout TZM-bl

expressing T cells at the late stage of diseases, our observation have implications for understanding of the diverse roles of IFN-induced proteins in different stages of HIV-1 infection and AIDS pathogenesis. 168 ELUCIDATING MECHANISMS BY WHICH MUTATIONS IN ENV CONTRIBUTE TO HIV-1 DRUG RESISTANCE Rachel Van Duyne , Phuong Pham, Jonathan Spindler, Ann Wiegand, Mary F. Kearney, Eric O. Freed NIH, Frederick, MD, USA Background: Despite the effectiveness of antiretroviral therapy (ART), virological failure can occur in HIV-1 infected individuals, often in the absence of recognized drug resistance mutations (DRMs). By performing in vitro selection experiments, we identified mutations within the HIV-1 envelope (Env) glycoprotein that broadly increase viral fitness by overcoming blocks to virus replication, including several selected in the presence of the antiretroviral (ARV) inhibitor Dolutegravir (DTG). The goal of this study was to determine the mechanism by which the Env mutations afford ARV escape. Methods: Virus replication and quantification of viral spread in the presence of ARVs were measured by propagating Env mutant viruses in a spreading infection in T-cell lines and primary PBMCs. Cell-free and cell-to-cell virus transmission was measured using reporter viruses and cell lines. Finally, we measured the effective multiplicity of infection (MOI) of viral transmission events. Results: We calculated the fold-change in IC 50 of two of the DTG-insensitive Env mutants, A556T and A539V, as 4-5 fold, comparable to that of current DTG DRMs in integrase. The de novo-selected DTG-resistant Env mutant, A539V, also exhibits markedly reduced sensitivity to at least two other classes of ARVs. Using a GFP-expressing reporter virus, we determined that the A539V mutation greatly enhances the efficiency of cell-to-cell transfer and increases the effective MOI of the transmitted virus. We are currently measuring the viral DNA load per infected cell in the presence and absence of DTG. Remarkably, we selected a DTG-resistant Env mutation at the same position in a subtype C transmitter founder virus. Finally, we observed that propagation of an ARV- resistant mutant in high concentrations of DTG forced selection of additional Env mutations, which may ultimately enable the acquisition of DRMs in integrase. Conclusion: These results provide insights into escape from ARVs and demonstrate that mutations in Env can contribute to broad HIV drug resistance in vitro. The study of Env mutants that result in a decreased sensitivity to DTG is of particular interest as resistance mutations in integrase have been challenging to characterize to date. We speculate that these Env mutations may provide a “stepping stone” on the path to high-level drug resistance in vivo.We are currently investigating the implications of these findings for HIV drug resistance in nonhuman primate models and in patients. 169 NONRANDOM GENERATION OF DELETIONS WITHIN HIV PROVIRAL SEQUENCES IN VIVO Bonnie Hiener 1 , Bethany A. Horsburgh 1 , Vincent Morcilla 1 , Eunok Lee 1 , Susanne von Stockenstrom 2 , Jeffrey M. Milush 3 , Teri Liegler 3 , Rebecca Hoh 3 , Rémi Fromentin 4 , Nicolas Chomont 4 , Steven G. Deeks 3 , Frederick M. Hecht 3 , Sarah Palmer 1 , Robert Lanfear 5 1 The Westmead Institute for Medical Research, Westmead, NSW, Australia, 2 Karolinska Institute, Stockholm, Sweden, 3 University of California San Francisco, San Francisco, CA, USA, 4 Université de Montréal, Montreal, QC, Canada, 5 Australian National University, Canberra, Australia Background: Most latent HIV proviruses integrated into CD4+ T cells of HIV positive individuals on long-term antiretroviral therapy (ART) are defective and replication-incompetent. The most common defects are deletions in the proviral sequence which are assumed to be generated by the same mechanism as HIV recombinants: template switching by the reverse transcriptase (RT) enzyme. Methods: To investigate whether deletions are generated by template switching, we 1) determined the distribution of deletion start and stop sites across the length of the HIV genome and compared this with known recombination sites, and 2) investigated whether the presence of nucleotide homology, a common feature of recombination sites also occurred between deletion start and stop sites. Near full-length HIV proviral sequences from CD4+ T cells from 10 participants on long-term ART (3-17 years) were obtained. Sequences containing internal deletions of ≥2 bases were identified and the position and sequence (surrounding 10 bases) of deletion start and stop sites recorded.

Poster Abstracts


CROI 2019

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