CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

cells that can mediate robust infection of CD4 + T cells in trans. Since CD169 expression is also induced by IFN-Is on macrophages, we hypothesized that CD169 induced by IFN-I could facilitate productive HIV-1 infection in macrophages in cis and thus offset antiviral effects of IFN-Is. Methods: To investigate the effect of IFN-Is on HIV-1 replication in myeloid cells, a monocyte cell line, THP-1 or primary monocyte-derived macrophages (MDMs) were treated with IFN α for 48 hours and infected with HIV∆env-luc reporter virus pseudotyped with HIV-1 Lai (HIV/Lai) or VSV-G (HIV/G) glycoproteins, or replication competent HIV-luc. HIV-1 fusion was measured by the conventional Vpr-BlaM assay. To investigate if CD169 + myeloid cells are productively infected in vivo, lymph nodes (LNs) from pigtailed macaques chronically infected with RT-SHIV mne027 were stained for p27 gag and CD169. Results: As reported previously, HIV/G infection was severely attenuated in IFN-treated-THP-1. Surprisingly, however, replication of HIV/Lai was enhanced in IFN-treated-THP-1 than in untreated THP-1. We found that HIV/Lai fusion was greatly enhanced in IFN-treated-THP-1, while that of HIV/G was severely attenuated. This enhanced fusion and infection depended on CD169 since pretreatment with α CD169 blocking antibody abrogated the enhancement of virus fusion and replication in IFN-treated-THP-1. IFN α treatment of MDMs also up-regulated CD169 and HIV-1 fusion in treated MDMs was enhanced (2-fold) in a CD169-dependent manner. Interestingly, CD169 enhanced virus replication in MDMs even in the presence of IFN α (>2-fold higher compared to MDMs pretreated with α CD169 blocking antibody). Finally, LNs from SHIV-infected macaques showing signatures of immune activation contained more CD169 + cells than those of uninfected animals and, intriguingly, a large proportion of p27 gag+ cells were also CD169 + . Conclusions: These studies suggest that HIV-1 has exploited CD169 to attenuate IFN-I-induced antiviral state in myeloid cells. 192 HIV and SIV Inhibition by RNA-Associated Early Stage Antiviral Factor (REAF) Aine McKnight Queen Mary University of London, London, United Kingdom Background: The interaction of viruses with their human host is a constant war. The discovery of novel anti-viral restriction factors illuminates unknown aspects of innate sensing and immunity. Methods: An siRNA screen of ~20,000 human genes was used to uncover those involved in inhibition of HIV replication. We identified RNA-associated Early-stage Anti-viral Factor (REAF) as an inhibitor of HIV replication Results: 114 genes were identified to be potentially involved in intrinsic resistance. Focusing on the most potent factors led us to REAF. REAF (previously RPRD2) was annotated in the human genome but with no known function. We observed more than 50 fold rescue of HIV-1 infection following knockdown of REAF by specific siRNA. Quantitative PCR was used to measure the effect of REAF knockdown on two steps in the replication cycle – production of reverse transcripts and integration of viral cDNA. Both steps were strongly enhanced. Conversely, when REAF is over expressed in target cells fewer reverse transcripts are produced. Human REAF can also inhibit HIV-2 and simian immunodeficiency virus (SIV) infection. REAF interacts (either directly or indirectly) with HIV RNA or RNA:DNA intermediates during reverse transcription. Also, during the process of reverse transcription REAF protein is degraded, within one hour of infection, in a proteosomal dependent manner. Furthermore, REAF can inhibit HIV replication via different routes of entry into cells. Its potency is, however, highly dependent on the pathway of entry used and we show it is the lentiviral restriction factor 2 (Lv-2) 1,2 . Conclusions: We propose that REAF is part of an anti-viral surveillance system destroying incoming retroviruses. This novel mechanism could apply to invasion of cells by any intracellular pathogen. 1 Marchant, D., Neil, S. J., Aubin, K., Schmitz, C. & McKnight, A. An envelope-determined, pH-independent endocytic route of viral entry determines the susceptibility of human immunodeficiency virus type 1 (HIV-1) and HIV-2 to Lv2 restriction. J Virol 79 , 9410-9418, (2005). 2 Schmitz, C., Marchant, D., Neil, S. J., Aubin, K., Reuter, S., Dittmar, M. T. & McKnight, A. Lv2, a novel postentry restriction, is mediated by both capsid and envelope. J Virol 78 , 2006-2016, (2004). 184 Regulation of the Innate Immune Sensing of HIV by the Viral Capsid and the Cytosolic DNA Sensor cGAS Nicolas Manel Institut Curie, Paris, France Background: Dendritic cells (DCs) play an important role in the detection of viral infections through innate sensing pathways and the induction of immune responses. The pathogenic virus HIV-1 escapes cytosolic innate immune sensing by monocyte-derived DCs. Innate sensing of HIV-1 by DCs can be rescued by complementing the virus with the small protein Vpx found in HIV-2, attributing the lack of cytosolic sensing of HIV-1 to to the poor ability of the virus to replicate in MDDCs as a result of the SAMHD1 restriction. In contrast, VSV-G-pseudotyped HIV-2 efficiently infects MDDCs and activates pathways of innate immunity. HIV-2 is much less pathogenic than HIV-1, suggesting that such recognition by DCs may be important for effective anti-HIV immune responses. However, the mechanism of cytosolic HIV sensing by DCs remained to be determined. Methods: Initial experiments showed that the viral capsid and its interaction with the cellular protein Cyclophilin A plays an important role. To study HIV sensing, we designed mutations in capsid to increase its affinity for Cyclophilin A (HIVac). Results: Strikingly, HIVac mutated viruses maintained the ability to activate DCs but had lost the ability to infect the DCs. Using such virus, we found that innate sensing requires synthesis of the viral cDNA, but not nuclear entry and genome integration. We also find that the wild-type HIV-1 capsid normally shields the cDNA before genome integration, preventing its detection by innate sensor(s). Finally, we examined cytosolic DNA sensors using RNAi. We find that the cytosolic DNA sensor cGAS (cyclic GMP-AMP synthase) is required for innate recognition of HIV-1 and HIV-2 cDNA by DCs. Conclusions: Altogether, these results establish that cGAS is an important innate sensor of HIV-1 and HIV-2 and uncover an essential regulation of this sensing mechanism by capsid and Cyclophilin A. We are currently investigating various aspects of these innate immune mechanisms and new findings will be presented. 203 Mapping Vpx and Vpr Specificity in Antagonism of SAMHD1 Chelsea Spragg ; Michael Emerman Fred Hutchinson Cancer Research Center, Seattle, WA, US Background: The lentiviral accessory protein Vpx enhances infectivity of macrophages, dendritic cells, and resting T-cells by inducing degradation of the restriction factor SAMHD1, which blocks replication at reverse transcription. Vpx bridges SAMHD1 to the host ubiquitin ligase substrate receptor DCAF1, leading to polyubiquitination of SAMHD1 and degradation by the proteasome. The vpx gene is present in only two major lineages of lentivirus including HIV-2, but the paralagous vpr is present in all extant lineages. In certain cases, Vpr also has the ability to degrade host SAMHD1. SAMHD1 has evolved under positive selection due to viral antagonism, resulting in species-specificity between host SAMHD1 and viral Vpx/r. Depending on the lineage, Vpx exclusively targets either the N-terminus or the C-terminus of SAMHD1; however, the regions of Vpx/r controlling specificity

Oral Abstracts

141

CROI 2015