CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

166 DETECTION AND CHARACTERIZATION OF CELLS EXPRESSING HIV RNA BY FLOW CYTOMETRY Aaron Christensen-Quick 1 , Amey Mukim 2 , Celsa A. Spina 2 , Sara Gianella 3 , Davey M. Smith 3 1 Univ of California San Diego, San Diego, CA, USA, 2 VA San Diego Hlthcare System, San Diego, CA, USA, 3 Univ of California San Diego, La Jolla, CA, USA

Background: Our current knowledge of HIV-infected cell populations is largely derived from flow cytometry studies using antibodies to p24- the HIV capsid protein and most abundantly expressed antigen. However, p24 is an imperfect marker of infected cells that requires artificial activation for its detection, especially when analyzing rare HIV+ cells from ART-treated individuals. Much less is known about cell populations that express both p24 and HIV RNA, despite their potential importance to cure efforts. Methods: Primary human CD4 T cells from HIV-negative blood donors were isolated by negative selection, activated with plate-bound α-CD3/CD28 and infected with HIV IIIB . Infected samples and uninfected controls were

analyzed by flow cytometry for their expression of immunological markers, HIV p24 and HIV RNA. Detection of HIV RNA was achieved using PrimeFlow™ probes covering the entire HIV genome. Significant differences in expression of lymphocyte markers were identified using the Wilcoxon rank test. Results: Results: In HIV-infected CD4 T cells from five donors, we readily detected cells that expressed HIV RNA, p24, or both (see figure). Importantly, a substantial population of cells from infected samples expressed HIV RNA, but not p24 (mean=2.7% of total cells). Over a third (mean=35%) of cells that expressed HIV RNA did not co- express p24. Except for background signal, these populations were absent in uninfected controls (mean<0.25% for RNA+p24-, <0.0001% for RNA+p24+, <0.06% for HIV RNA- p24+, n=3). HIV-infected samples, but not uninfected controls, had a population of cells expressing decreased CD45RA (1.9% vs 0.005%, p<0.02), and this population of CD45RA low cells was enriched in both HIV RNA+p24+ cells (29.4% vs. 5.2% of total, p<0.008) and RNA+p24- cells (9.9% vs. 2.7% of total, p<0.03). Interestingly, the average percent of CD27+ cells was significantly lower in HIV RNA+ p24- cells from infected samples, but not in HIV RNA+ p24+ dual-positive cells (p<0.05 and p=.17, respectively). Conclusion: These findings emphasize the need for a more detailed characterization of cells supporting HIV transcription. Differences between cells expressing HIV RNA and those producing p24 may have important biological consequences, especially in regard to HIV persistence.

Poster and Themed Discussion Abstracts

167 INFLAMMASOME-REGULATED CYTOKINE IL-37 INHIBITS HIV-1 REPLICATION IN PRIMARY HUMAN CD4 Neel Sangal, Guido Massaccesi, Jeffrey Quinn, Andrea Cox, MatthewWinter Johns Hopkins Univ, Baltimore, MD, USA

Background: Interleukin (IL)-37 is a recently characterized member of the IL-1 family of cytokines that regulate innate immune responses. Like other inflammasome- regulated cytokines it’s maturation and release is dependent on inflammasome activity and caspase-1 activation. HIV-1 infected individuals have elevated levels of circulating inflammasome cytokines. Methods: We assessed the effects of inflammasome-regulated cytokines on HIV-1 replication in human primary CD4+ T-cells. CD4+ T-cells were sorted from human blood and infected with HIV RF or IIIB followed by culture with cytokines in various concentrations. Viral replication was measured by p24 ELISA and viral RNA. Cytokine receptor expression was assessed by flow cytometry. Modulation of innate immune genes was determined by qRT-PCR. Results: We find that activated T-cells express receptors for all inflammasome cytokines; however only IL-37 exerts an antiviral effect in HIV-1 infection. IL-37 induces expression of antiviral factors including IFITM proteins and APOBEC3A in CD4+ T-cells. IL-37 directly inhibits HIV-1 replication in infected primary human CD4+ T-cells by inhibiting life cycle steps prior to integration of the pro-viral genome. Conclusion: IL-37 exerts a suppressive effect on HIV-1 by inducing expression of innate antiviral factors known to restrict HIV replication. 168 BCA2 INTERFERES WITH HIV-1 TRANSCRIPTION BY ENHANCING THE SUMOYLATION OF IΚBΑ Marta Colomer-Lluch Texas Tech Univ, Lubbock, TX, USA Background: BCA2 (breast cancer-associated gene 2) is an E3 ubiquitin ligase that serves as a co-factor in the restriction imposed by Tetherin on HIV-1. We recently demonstrated that BCA2 also has Tetherin-independent activity. Particularly, BCA2 targets HIV-1 Gag for lysosomal degradation, impairing virus assembly. Since many antiviral factors modulate the NF-κB pathway, we sought to explore if BCA2 is harnessing this innate cascade to further limit HIV-1 Methods: To study the role of BCA2 on NF-κB, gene expression and NF-κB-inducible luciferases assays were performed. Results obtained from these studies were corroborated by depleting cells from BCA2 and its co-factors using shRNAs. To examine the implication of the BCA2-mediated regulation of NF-κB in HIV-1 infectivity, replication and transcriptional assays were conducted in CD4+ cells. To identify the molecular mechanism by which BCA2 modulates this pathway, the post-translational modifications of NF-κB components were analyzed as well as their subcellular distribution Results: Here we show that BCA2 is induced by NF-κB-activating cytokines and its up-regulation provides a negative feedback on NF-κB. Knockdown assays revealed that UBC9 is a critical BCA2 co-factor to inhibit the NF-κB pathway. UBC9 mediates the SUMOylation of IκBα, which in turn impairs the nuclear translocation of NF-κB. To explore if BCA2 participates in this process, we assessed IκBα’s post-translational modifications. Remarkably, the levels of SUMOylated IκBα increase in cells overexpressing BCA2 (2-fold) whereas its phosphorylation levels diminish (10-fold). Conversely, depletion of UBC9 or BCA2 leads to a significant reduction of SUMOylated IκBα and a corresponding increase in its phosphorylation levels. In vitro SUMOylation studies revealed that BCA2 enhances IκBα SUMOylation, demonstrating for the first time that BCA2 serves as a SUMO-ligase in the regulation of the NF-κB pathway. Consistent with this, BCA2 blocks the nuclear translocation of NF-κB. Since HIV-1 needs NF-κB to enhance its replication, we studied the biological implication of the BCA2-dependent inhibition of this pathway in HIV-1 infectivity. BCA2 reduces HIV-1 transcription up to 4-fold in CD4+ cells. However, HIV-1 partially circumvents this hurdle by decreasing the steady-state levels of BCA2 via a yet unidentified mechanism Conclusion: BCA2 poses several barriers to HIV-1 infection: not only does BCA2 prevent assembly and release of nascent virions, but also restricts HIV-1 at the transcriptional level 169 RHO FAMILY GTPASES ENHANCE HIV-1 INFECTION Mark Lucera 1 , Zach Fleissner 2 , Caroline Tabler 2 , Daniela Schlatzer 2 , Zach Troyer 2 , John Tilton 2 1 Univ of Colorado, Aurora, CO, USA, 2 Case Western Reserve Univ, Cleveland, OH, USA Background: HIV-1 co-opts host cellular machinery to complete its replication cycle, including cytoskeletal components for intracellular trafficking, nucleoproteins for pre- integration complex (PIC) import, and the ESCRT pathway for assembly and budding. It is widely recognized that cellular post-translational modifications (PTMs) regulate protein activity within cells; however, little is known about how PTMs influence HIV replication. Previously, we reported that blocking deacetylation of tubulin using histone deacetylase (HDAC) inhibitors promoted the kinetics and efficiency of early post-entry viral events. To uncover additional PTMs that modulate entry and early post-entry stages in HIV infection, we employed a flow cytometric approach to assess a panel of small molecule inhibitors on viral fusion and LTR promoter-driven gene expression.

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CROI 2017