CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

POSTER ABSTRACTS

TUESDAY, FEBRUARY 24, 2015 Session P-A1 Poster Session

Poster Hall

2:30 pm– 4:00 pm Cellular Innate Immunity

183 STAT5 Inhibition Reduces HIV-1 Infection and TLR7/8 Responses in Human Macrophages Sofia Appelberg 1 ; Carla N. Mavian 1 ; Julie C.Williams 1 ; Philip Lichlyter 1 ; John Sleasman 2 ; Maureen M. Goodenow 2 1 University of Florida, Gainesville, FL, US; 2 Duke University, Durham, NC, US

Background: Persistent production of HIV-1 by cellular reservoirs and chronic immune activation are major challenges in the elimination of infection and in the health of HIV-1 infected individuals. Monocyte derived macrophages (MDM) are significant contributors to continual viral production and chronic inflammation. HIV-1 infection of macrophages activates STAT1, STAT3 and STAT5. Infection also primes MDM rendering the cells hypersensitive to subsequent TLR stimulation. STAT5 has two functional binding sites in the HIV-1 LTR, although the effects of STAT5 activation on HIV-1 infection and immune responses by MDM are unknown. We tested the hypothesis that STAT5 activation supports HIV-1 infection and priming of MDM. Methods: Human MDM or TZM-bl cells were treated with a STAT5 specific inhibitor or transfected with STAT5a/b siRNA prior to infection with replication competent HIV-1 AD or single cycle, luciferase-tagged HIV-1 JRFL envelope pseudotyped virus. Inhibition of STAT5 protein was verified by western blots. Infection was monitored by measuring supernatant p24 levels, luciferase enzyme activity, non-integrated gag and integrated HIV-1 copies. Cell surface expression of CD4 and CCR5 or intracellular p24 and TLR3, TLR4 or TLR7/8 induced TNF and IL-6 was determined by flow cytometry or ELISA. Results: Pharmacologic inhibition or siRNA-mediated knockdown of STAT5 significantly reduced HIV-1 infection of MDM compared to control. Suppression of de novo or established infection by STAT5 inhibition was independent of changes in CD4 or CCR5 surface expression or number of non-integrated gag or integrated HIV-1 copies, but related to a significant reduction in HIV-1 LTR activity. STAT5 inhibition dramatically reduced TNF and IL-6 production mediated specifically through TLR7/8, but not through TLR3 or TLR4, in both infected and uninfected cells. Conclusions: Our novel findings show a direct and indirect role for STAT5 in modulating HIV-1 infection in MDM. STAT5 directly support transcription of the viral genome, with no effect on host cell expression of CD4 or CCR5 and steps in the viral life cycle from entry through integration. STAT5 is involved in pro-inflammatory cytokine production induced by the TLR7/8 receptors, which sense ssRNA and viral infections, indicating an indirect role in HIV-1 infection. Disruption of TLR7/8 signaling mediated by STAT5 inhibition provides no 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. 185 cGAS Induced Type I IFN Responses in Dendritic Cells FromHIV Elite Controllers Enrique Martin-Gayo 1 ; Jacqueline Cronin 1 ; Zhengyu Ouyang 1 ;Taylor Hickman 1 ; JohnTrombetta 2 ; Florencia Pereyra 1 ; BruceWalker 1 ; Alex Shalek 1 ; Mathias Lichterfeld 3 ; XuYu 1 1 Ragon Institute of MIT, MGH and Harvard, Boston, MA, US; 2 MIT Institute for Medical Engineering & Science, Boston, MA, US; 3 Massachusetts General Hospital, Boston, MA, US Background: Cell-intrinsic HIV-1 immune recognition by IFI16 in CD4 T cells can cause pyroptosis, CD4 T cell loss, and increased immune activation. However, such cell-intrinsic immune responses in alternative cell subsets are thought to contribute to protective immune activity against HIV-1. Indeed, our previous studies indicated an accumulation of viral reverse transcripts and a rapid and sustained cell-intrinsic type I IFN response in primary conventional dendritic cells (cDC) from Elite controllers (EC) upon HIV-1 infection, which was functionally relevant for inducing and supporting effective HIV-1-specific CD8 T cell responses. However, molecular mechanisms accounting for such efficient cell-intrinsic immunity against HIV in cDCs from these patients remain unknown. Methods: cDCs from EC, untreated chronic progressors (CP), HAART-treated and HIV-1 negative subjects were ex vivo infected with HIV-1, followed by assessments of expression changes of putative cytoplasmic DNA sensors. siRNA-mediated gene silencing was used to identify host factors responsible for the induction of cell-intrinsic immune responses. Single-cell RNA-Seq analysis of HIV-1-infected cDC was conducted to indentify gene expression pathways associated with cell-intrinsic HIV-1 immune recognition in cDC. apparent advantage for the virus. Thus, STAT5 is a key factor for HIV-1 replication and a novel anti-viral drug candidate. 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

Poster Abstracts

197

CROI 2015