CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

Methods: A panel of small molecule inhibitors tailored to enzymes involved in epigenetic and PTM pathways were analyzed for effects on HIV fusion and gene expression by flow cytometry. Compounds with ±0.5 log2-fold change effects on viral infection were validated in additional experiments using CD4+ T cells from 5 healthy controls. Additionally, a phosphoproteomic experiment was performed to identify signaling pathways altered by HIV binding to CD4 and CCR5. Results: While viral fusion was not significantly affected, early post-entry viral events were modulated by drugs targeting multiple processes including histone deacetylation, methylation, and bromodomain inhibition. Most notably, we observed that inhibitors of the Rho GTPase family of cytoskeletal regulators – including RhoA, Cdc42, and Rho- associated kinase (ROCK) signaling pathways – significantly reduced viral infection. Proteins in the Rho GTPase signaling cascades were also identified using a phosphoproteomic analysis of virion-induced signaling via CD4 and CCR5, suggesting that HIV-1 initiates the manipulation of the host cytoskeletal network via interactions with CD4 and CCR5 at the cell surface to promote its replication. Conclusion: Together, these data provide evidence that the Rho GTPase family of cytoskeletal regulators play a major role in facilitating HIV infection and suggest that the virus actively modulates the cytoskeleton by signaling through CD4 and CCR5. 170 SEMEN FACTORS ASSOCIATED WITH SEMEN VIRAL LOAD AND INFECTION-ENHANCING ACTIVITY Christopher D. Pilcher 1 , Kara Marson 1 , Peter Bacchetti 1 , Sheila Keating 2 , Wendy Hartogensis 1 , Teri Liegler 1 , H. Ewa Witkowska 1 , Satish K. Pillai 2 , Frederick M. Hecht 1 , Nadia R. Roan 3 1 Univ of California San Francisco, San Francisco, CA, USA, 2 Blood Systems Rsr Inst, San Francisco, CA, USA, 3 Gladstone Inst of Virology, San Francisco, CA, USA Background: We have previously reported that seminal plasma from HIV negative men can markedly enhance HIV infection of permissive cells, and that this activity is positively associated with endogenous concentrations of SEM1(86-107), a peptide derived from a protein produced by the seminal vesicles. In vitro, SEM1(86-107) polymerizes to form amyloid fibrils that enhance HIV infection, and endogenous SEM1(86-107) amyloid aggregates can be detected in semen. We hypothesized that in HIV-infected individuals, semen factors that promote HIV infection might also increase the semen HIV viral load. Methods: We examined the relationships among semen viral load, HIV infection enhancing activity, and inflammatory cytokines in semen samples from 122 HIV-infected individuals. Samples were obtained at least 4 months post seroconversion; 149 specimens were obtained from 103 ART naïve donors, 20 donated pre-and post-ART suppression, and 20 pre-and post-interruption of ART. HIV infection of TZM-bl cells in the presence vs. absence of semen was used to quantitate viral enhancing activity. Inflammatory cytokines and chemokines were measured by Luminex in semen and blood. Results: The degree to which semen enhanced HIV infection varied (from no enhancement to 30-fold) between HIV-infected individuals but was stable over time within individuals. Higher infectivity enhancement of semen was strongly correlated with the concentration of SEM1(86-107) in HIV-infected men (r=0.70, p<0.0001). In multivariate analyses controlling for the blood viral load, higher semen viral load was independently associated with higher semen infectivity enhancing activity (p=0.02) as well as with higher levels of IL8 (p=0.01) and IL6 (p=0.01). Mean IL8 levels were 245-fold higher and mean IL6 levels 25-fold higher in semen than in blood. In longitudinal experiments with patients initiating and interrupting ART, we found that changes in semen viral load with ART use did not substantially alter levels of infectivity enhancement, SEM1(86-107), or pro-inflammatory cytokines in semen. Conclusion: The composition of semen can differ greatly between HIV-infected men. Some of the factors that vary between men, such as infection-enhancing amyloids and certain proinflammatory cytokines, may facilitate HIV transmission in two ways-by promoting local viral replication in the genital tract (increasing semen viral load), and/or by enhancing infection of mucosal target cells. 171LB STRUCTURAL BASIS FOR INHIBITOR-INDUCED AGGREGATION OF HIV INTEGRASE Kushol Gupta 1 , Vesa Turkki 1 , Scott Sherrill-Mix 1 , Young Hwang 1 , Grant Eilers 1 , Robert T. Nolte 2 , Emile Velthuisen 2 , Jerry Jeffrey 2 , Gregory Van Duyne 1 , Frederic Bushman 1 1 Univ of Pennsylvania, Philadelphia, PA, USA, 2 Glaxosmithkline, Rsr Triangle Park, NC, USA Background: The allosteric integrase (IN) inhibitors (ALLINIs) target the viral-encoded IN protein and interfere with HIV replication via disruption of viral particle assembly late during HIV replication. Methods: To investigate their inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264, and determined the structure of the complete inhibitor interface at 4.4 Å resolution. Results: In this structure, GSK1264 is buried between the catalytic core domain of one IN dimer and the CTD of an adjacent IN dimer. The amino-terminal domain is not resolved in the structure but does not participate in GSK1264 binding. The GSK1264 binding interface is rich in residues implicated in IN oligomerization and ALLINI sensitivity, indicating likely functional significance. The IN-IN interaction mediated by GSK1264 leads to formation of an open polymer in the crystal, a polymerization reaction that is readily reproduced in solution with purified components. To probe ALLINI function more broadly, we compared the properties of several ALLINIs in biochemical, biophysical, virological, and electron microscopic assays. Several ALLINI escape mutations encode IN substitutions at or near the inhibitor binding site, and these also resulted in decreased IN oligomerization in vitro. The results support a mechanismwhere ALLINIs disrupt viral particle maturation by promoting formation of the IN polymers observed in the IN-GSK1264 crystal structure. Additionally, the results support a structural model for the catalytically inactive IN tetramer discussed in several previous studies. Identification of the interface responsible for polymer formation provides data useful for improving HIV inhibitors and helps explain a wealth of previous studies of HIV IN.

Poster and Themed Discussion Abstracts

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CROI 2017