CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

Results: Buprenorphine increased HIV-1 infectivity at the three MOIs tested (0.1, 0.2 and 0.5) and over all tested periods (24, 48 and 72 hours; n=3 experiments). For example, at 72 hours, HIV infection was three to six times higher in the presence of 2 nM buprenorphine (p<0.0001). Morphine also enhanced HIV-1 infection, but to a much lesser extent and only at the higher dosage. At 100 μM, morphine caused a 1.4-fold increase of infection after 72 hours (p<0.0001), whereas at 1 µM, HIV-1 infectivity remained unchanged. In none of the leukocyte types tested, we found expression of the classical opioid receptors (mu, kappa or delta), but the nociception/orphanin FQ receptor (NOP also known as OPRL1) was present in blood and vaginal lymphocytes. Conclusion: Our results suggest that buprenorphine, much more than morphine, increases the susceptibility of leukocytes to HIV-1 infection in vitro. Given that leukocytes do not express the classical opioid receptors, this increase in HIV susceptibility must occur through either OPRL1 or an unknown pathway. These findings are a first step toward understanding how opioids, including those used for MAT, affect HIV infection. 182LB EBV BORF2 INHIBITS AND SEQUESTERS HUMAN APOBEC3B TO PROTECT VIRAL GENOME INTEGRITY Adam Cheng 1 , Jaime Yockteng Melgar 2 , Lori Frappier 2 , Reuben S. Harris 1 1 University of Minnesota, Minneapolis, MN, USA, 2 University of Toronto, Toronto, ON, Canada Background: Human cells express up to seven different DNA cytosine deaminases, APOBEC3(A3)A-H, which have overlapping functions in innate antiviral immunity. Several A3s, most notably A3G, have been well characterized in inhibiting HIV-1 replication by converting C-to-U in retroviral single- stranded cDNA replication intermediates, thereby generating hypermutated viral genomes. To overcome A3-mediated restriction, viruses have evolved counteraction measures such as the lentiviral Vif protein, which nucleates the formation of a ubiquitin ligase complex that degrades A3 enzymes. We hypothesize that, like HIV-1 and related lentiviruses, herpesviruses including Epstein-Barr virus (EBV) possess mechanisms to counteract restriction by A3 enzymes. Methods: The EBV ribonucleotide reductase subunit BORF2 was used in proteomic experiments to identify cellular binding partners. Interactions were confirmed using co-IP and mutagenesis experiments. Recombinant proteins were used in DNA C-to-U assays. Immunofluorescence and live cell imaging were used to address subcellular localization. CRISPR was used to generate BORF2- null EBV, and Sanger and next-generation sequencing were used to quantify viral genome integrity during lytic reactivation. Results: The BORF2-APOBEC3B (A3B) interaction was discovered in a proteomic screen and validated by immunoprecipitation experiments and binding assays with recombinant proteins. Structure-guided mutagenesis experiments map the interaction to a conserved loop in the A3B catalytic domain, and biochemical studies demonstrate stoichiometric inhibition of A3B DNA cytosine deaminase activity by BORF2. Fluorescence and electron microscopy images, including movies of full cell division cycles, reveal a dramatic BORF2-dependent re-localization of nuclear A3B to perinuclear foci that may be residual bodies. CRISPR/Cas9-mediated deletion of BORF2 renders EBV susceptible to hypermutation by A3B. Conclusion: The large double-stranded DNA herpesvirus EBV uses the large subunit of its ribonucleotide reductase, BORF2, to counteract A3B-mediated restriction/mutagenesis in a two-pronged approach through direct enzymatic inhibition and cellular relocalization. This mechanism is distinct from the proteasomal degradation mechanism used by lentiviruses and suggests new avenues for intervention. 183 R5X4 EVOLVE DISTINCT GP41 ACTIVATION PATHWAYS TO SENSE ALTERNATE CORECEPTOR BINDING Annemarie Laumaea 1 , Benjamin K. Dickerman 1 , Jacqueline K. Flynn 1 , Paul R. Gorry 2 , Jason M. Mackenzie 3 , Heidi E. Drummer 1 , Pantelis Poumbourios 1 1 Burnet Institute, Melbourne, VIC, Australia, 2 RMIT University, Melbourne, VIC, Australia, 3 University of Melbourne, Melbourne, VIC, Australia Background: Entry of HIV-1 is mediated by its envelope glycoproteins gp120 and gp41 (Env) that exist as a non-covalently associated trimer of heterodimers on the surface of virions. gp120 forms the outer layer and initial binding to its primary receptor CD4 creates a binding site for one of two obligatory chemokine co-receptors (CKR), CCR5 or CXCR4. CKR binding triggers refolding of gp41, enabling six-helix bundle formation that necessitates pore formation and

transfer of viral core to the cell interior. gp120’s interaction with CCR5 and/ or CXCR4 classifies Env into R5 or X4 phenotypes, respectively. Transmitted/ founder viruses that establish infection in a new host use CCR5 while X4 emerge and predominate in late stage disease in a subset of infected individuals. R5X4 isolates use both CKRs, emerge prior to the acceleration of T cell decline and the onset of symptomatic disease, and are believed to represent an evolutionary intermediate between R5 and X4 isolates. The molecular determinants that control CKR binding by R5 and X4 gp120 molecules is well defined however, downstream events that lead to the activation of the fusion function of gp41 is still unclear. We aimed to examine how receptor-induced signals by R5X4 Envs, are transmitted to activate gp41. Methods: Near neighbour contacts within the gp120-gp41 association interface were identified by in silico structural screen and conservative substitutions were introduced to these sites in the primary R5X4 Env of the brain derived strain, dBR07. The ability of the mutant viruses to use alternative CKRs was assessed in a TZM-bl infectivity assay and subsequently in primary CD4+ T cells and PBMCs. Results: Three gp41 mutants were identified with differential abilities to mediate infection via CCR5 versus CXCR4: F522Y in the FP and K574N and Q591A in heptad repeat 1. K574 and Q591 were critical for CCR5-mediated infection of PBMCs, primary CD4+ T cells and U87.CD4.CCR5 but dispensable for CXCR4- mediated infection. F522, however was essential for CXCR4-mediated infection of U87.CD4.CXCR4 cells, whereas infectivity for U87.CD4.CCR5 cells was retained. Interestingly, F522Y was not infectious for cells with low CCR5 expression in the presence of CCR5 antagonists. Importantly, K574N and Q591A retained their X4 phenotypes in the context of other R5X4 Envs. Conclusion: The data suggest that R5X4 evolve distinct gp41-fusion activation pathways that sense alternative CKRs binding, and may represent a key step in the evolution of R5X4 and ultimately X4 HIV-1.

Poster Abstracts

184 VARIABILITY OF HIV-1 V2 ENV DOMAIN FOR INTEGRIN BINDING: CLINICAL CORRELATES Claudia Fabrizio , Davide F. Bavaro, Luciana Lepore, Antonella Lagioia, Gioacchino Angarano, Laura Monno, Annalisa Saracino University of Bari, Bari, Italy Background: The HIV V2 179-181 (HXB2 numbering) tripeptide mediates binding to α4β7 integrin, which is responsible for GALT homing. The exact role of V2 in viral transmission and replication and possible clinical correlates are still unclear. We aimed to assess V2 variability in naive HIV-1 infected patients and its association with clinical and viro-immunological features. Methods: Gp120 sequences were obtained from 323 subjects newly diagnosed with HIV infection; V2 regions were evaluated for length, potential N-linked glycosylation sites (PNGs), net charge (NC) and tripeptide motif at residues 179-181. A possible association with HIV subtype, R5/X4 co-receptor tropism (CRT) (FPR 10%), duration of HIV infection (based on ambiguous nucleotides frequency in RT/PR, e.g. ≤0.2% ambiguity=recent infection) and patients’

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CROI 2018