CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

43 PQBP1 Is a Retrovirus-Specific Sensor Mediating cGAS/IRF3-Dependent Innate Responses Sunnie M. Yoh 1 ; Monika Schneider 1 ; Stephen Soonthornvarcharin 1 ; Rana Akleh 1 ; Kevin Olivieri 1 ; Paul De Jesus 1 ; Chunhai Ruan 2 ; Elisa de Castro 3 ; Pedro Ruiz 1 ; Adolfo Garcia-Sastre 3 1 Sanford-BurnhamMedical Research Institute, La Jolla, CA, US; 2 University of Michigan, Ann Arbor, MI, US; 3 Icahn School of Medicine at Mount Sinai, New York, NY, US Background: Infection of dendritic cells (DCs) by HIV-1 triggers an innate immune response that has been shown to be mediated through a cyclic GAMP synthase (cGAS)/IRF3- dependent pathway. We hypothesized that this pathway was activated by a cellular factor that recognizes a retrovirus-encoded molecular feature upstream of cGAS/IRF3. In an attempt to identify this proximal sensor of a HIV-1 pathogen-associated molecular pattern (PAMP), we performed a targeted RNAi screen utilizing primary human monocyte- derived DCs (MDDCs) and identified the protein PQBP1 as an innate immune sensor for HIV-1 DNA in myeloid cells. Methods: We utilized human MDDCs to perform a sub-genomic RNAi screen of putative nucleic acid-binding proteins to identify cellular factors required for the innate response to HIV-1 infection. Screening “hits” were further assayed for their ability to directly associate with HIV-1 nucleic acids in infected MDDCs. PQBP1 was the only molecule found to be required for both innate signaling and that associated with HIV-1 nucleic acids. PQBP1 was subjected to extensive validation using reconstituted stable shRNA and CRISPR cell lines, measurement of cellular cGAMP levels, in vivo and in vitro DNA binding assays and characterization of protein interactions. Results: Through our analysis, we demonstrated that PQBP1 directly binds to reverse transcribed HIV-1 DNA, and that it interacts with cGAS to initiate an IRF3-dependent innate response in myeloid cells. Further analysis revealed that PQBP1 associates with HIV-1 DNA through its C-terminus, but that it requires an intact WW-domain to associate with cGAS. Our study indicates that PQBP1 specifically recognizes a restricted set of retrovirally encoded DNA PAMPs, possibly originating from the secondary structure of RT intermediates. Conclusions: The ability to recognize invariant microbial features, and not pathogen specific elements, such as DNA sequence, is the definition of a pattern recognition receptor (PRR), and our evidence suggests that PQBP1 falls into this category. Our study presents a molecular understanding of how HIV-1 is recognized by professional antigen presenting cells to trigger cell-autonomous antiviral responses to promote adaptive immunity. Identification of PQBP1 as a direct sensor of retroviral DNA provides an opportunity for development of pharmacological agonists that may potentially improve HIV vaccine efficacies. 44 Mucosal HIV-1 Transmission Specifically Selects for Type 1 Interferon-Resistant Viruses Shilpa Iyer 1 ; Frederic Bibollet-Ruche 1 ; Christiana M. Shaw 1 ;Weiyu Zhang 1 ;Yingying Li 1 ;Timothy Decker 1 ; George M. Shaw 1 ; Persephone Borrow 2 ; Beatrice H. Hahn 1 1 University of Pennsylvania, Philadelphia, PA, US; 2 University of Oxford, London, United Kingdom Background: Mucosally transmitted founder (TF) HIV-1 are more resistant to the antiviral effects of type 1 interferons (IFNs) than viruses that predominate during chronic infection. To determine whether IFN-resistant viruses are specifically selected for during HIV-1 transmission, we derived viral isolates by limiting dilution from both plasma and genital fluids of chronically infected transmission donors and examined their relative IFN resistance. Methods: Plasma and cervicovaginal lavage (CVL) were collected from the donors of epidemiologically linked transmission pairs. Plasma or the non-cellular fraction of CVL samples were incubated with activated CD4+ T-cells to generate limiting-dilution isolates. Half genome sequences were analyzed to characterize the extent of genetic diversity. IFN α sensitivity of each isolate was analyzed by determining the IC50 in primary CD4+ T-cells. Results: We first used plasma from patient CH212 to determine that 500-1000 vRNA copies were required to obtain limiting dilution isolates. The diversity among 18 CH212 isolates was 2.5%, comparable to that observed by SGS analysis of the plasma vRNA. We determined the IFN α sensitivity of each virus isolate. IC50 values ranged from 8 to 115 U/ ml and were significantly lower than the IC50 value of the TF virus (240 U/ml) of the CH162 recipient. In an attempt to isolate IFN α -resistant viruses from the plasma, the limiting dilution isolation method was repeated in the presence of IFN α . The IFN α IC50 values of the 5 new isolates ranged from 155 to 225 U/ml, revealing that IFN α resistant viruses are present, but infrequent, in the donor plasma. We next obtained limiting-dilution isolates from plasma (n=10) and matching CVL (n=10) from donor CH492. The mean genetic diversity and the mean IFN α IC50 values were comparable between isolates derived from plasma and genital secretions. Interestingly, one CVL-derived isolate had a significantly higher IFN α IC50 value and was genetically most closely related to the single TF identified in the CH427 recipient. Conclusions: Viruses circulating in plasma and sexual secretions of chronically infected individuals exhibit a wide range of IFN resistance, and are generally more IFN sensitive than TF viruses that establish de novo infections. However, some donor viruses exhibit elevated resistance to IFN α and seem to be those that are preferentially transmitted. Thus, IFN-resistant viruses are specifically selected from a pool of biologically diverse viruses during mucosal HIV-1 transmission. 45 The Dynamics of HIV-1 RNA Near the Plasma Membrane During Virus Assembly Luca Sardo ; Steven C. Hatch; Jianbo Chen; Olga A. Nikolaitchik; Ryan C. Burdick; De Chen; Christopher J.Westlake; Stephen Lockett;Vinay K. Pathak;Wei-Shau Hu Frederick National Laboratory for Cancer Research, Frederick, MD, US Background: HIV-1 must package its RNA genome to generate infectious particles. The interactions between viral RNA elements and the structural protein Gag are essential for efficient virus particle assembly. However, little is known about the dynamics and the events that lead to RNA packaging in living cells. We investigated 1) the window of opportunity for HIV-1 RNAs near the plasma membrane to interact with Gag and be packaged into viral particles, and 2) quantified the proportion of HIV-1 RNAs that, after reaching the plasma membrane, were packaged into assembling viral particles. Methods: We labeled HIV-1 RNA with an RNA-binding protein fused to EOS, a photoconvertible protein whose fluorescence switches from green to red upon exposure to near-UV light. We selectively converted EOS near the plasma membrane of HeLa cells and followed the RNA signals over time by using total internal reflection fluorescence (TIRF) microscopy. Results: In the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, with ~50% of the RNAs departing within ~2 minutes; however, a portion of the RNAs (~20%) were detectable 30 minutes later. The presence of HIV-1 Gag increased the RNA retention time near the plasma membrane; most of the RNAs (~60%) were detected after 30 minutes. By tagging HIV-1 Gag with a blue fluorescent protein we observed that in the early phase of HIV-1 expression, when few or no Gag puncta are detected, ~10-40% of the HIV-1 RNAs that reached membrane were recruited into assembling particles. Conclusions: These studies are the first measurements of HIV-1 RNA dynamics at the plasma membrane and the efficiency of RNA recruitment, which provide insights into the events leading to the generation of infectious HIV-1 virions. 46LB Mechanisms of Dendritic Cell-Mediated Transfer of HIV-1 to CD4 + T Lymphocytes

Oral Abstracts

Mickael M. Menager 1 ;Wendy Lin 1 ; Jarrod S. Johnson 2 ; Kristen Dancel-Manning 1 ; Nicolas Manel 3 ; Feng-Xia Liang 1 ; Dan R. Littman 1 1 Skirball Institute of Biomolecular Medicine, New York, NY, US; 2 Seattle Biomedical Research Institute, Seattle, WA, US; 3 Institut Curie, Paris, France

Background: Dendritic cells (DCs) have essential roles in early detection of pathogens and in subsequent activation of both innate and adaptive immune responses, and are likely to be of critical importance in developing new strategies for protective HIV-1 vaccination. Whereas human DCs are resistant to productive viral replication, they have a unique ability to take up virus and transmit it efficiently to T lymphocytes. By doing that, the virus may evade, at least in part, the first line of defense of the immune system in mucosal tissues, exploiting DCs instead to facilitate rapid infection of a large pool of immune cells.

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CROI 2015