CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

support a strategy to disrupt Nef dimerization by small molecules as a new path to antiretroviral drug discovery. 189 RESTING HIV-INFECTED CD4 T CELLS EXPRESS NEF AND VPU, WHICH DOWNREGULATE MHC AND BST2 Rodrigo Matus-Nicodemos , Daniel Douek, Richard A. Koup National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA Background: The HIV reservoir resides in resting memory CD4 T cells. These infected cells are difficult to eradicate because of their lack of virus expression. Current eradication strategies aim to reactivate virus expression in these cells, allowing them to be recognized and killed by immune CD8 T cells specific for HIV peptide/MHC-I complexes (pMHC). In addition, broadly neutralizing antibodies may bind to cell surface Env protein and elicit antibody-mediated killing. However, HIV encodes two proteins, Nef and Vpu, which allow escape from both these eradication strategies through downregulation of pMHC and tetherin (BST2). Importantly, the timing of the expression of Nef and Vpu in HIV-infected resting CD4 T cells remains unclear. Methods: We aimed to explore this aspect of HIV infection by direct infection of resting CD4 T cells with two CCR5-tropic replication-competent GFP reporter viruses. For one virus GFP reports the expression of the Nef transcript, and for the other virus GFP reports the expression of the Vpu/Env transcript. We sorted CD4 T cells that were lacking expression of four activation markers: CD69, CD25, CD154 and HLA-DR. These resting CD4 T were infected with either reporter virus and examined daily for the expression of GFP, cell surface CD4, HLA-A02/-B07, tetherin, CD45RO, and CCR5 by antibody staining. Env expression was monitored by staining with the broadly neutralizing antibodies PG9 or VRC07. Results: Our data show that HIV directly infects resting memory CD4 T cells expressing CCR5. GFP expression for either virus starts to appear 3 to 4 days after infection. The GFP-positive cells infected with either virus showed downregulation of CD4, HLA A02, HLA-B07, and tetherin. These resting HIV- infected cells lacked the surface expression of Env and did not express infectious virions by virus outgrowth assays from the supernatant. In addition, TCR activation of these HIV-infected CD4 T cells attenuated the upregulation of both pMHCs and BST2. Conclusion: We conclude that HIV directly infects resting memory CD4 T cells to establish the reservoir. This direct infection of resting memory CD4 T cells confers a replicative advantage to HIV by expressing Nef to downregulate pMHC and Vpu to downregulate BST2 before activation for virion production. We therefore believe latently HIV-infected cells are cloaked from recognition by the immune system, thus providing a new strategy for persistence. 190 IMPAIRED NEF’S ABILITY TO COUNTERACT SERINC5 BY IMMUNE-DRIVEN MUTATIONS Mako Toyoda 1 , Doreen Kamori 2 , Jonathan Carlson 3 , Ai Kawana-Tachikawa 4 , Hiroyuki Gatanaga 5 , Shinichi Oka 5 , Takamasa Ueno 1 1 Kumamoto University, Kumamoto, Japan, 2 Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania, United Republic of, 3 Microsoft Research, Redmond, WA, USA, 4 National Institute of Infectious Diseases, Tokyo, Japan, 5 National Center for Global Health and Medicine, Tokyo, Japan Background: The host proteins SERINC 3 and 5 (SERINC3/5) are inhibitors of HIV-1 infectivity that are counteracted by Nef. Introduction of mutations to the highly conserved FPD 121-123 motif in Nef resulted in disruption of Nef›s ability to counteract SERINC5 and enhance infectivity. Because this region encompasses HLA-restricted CTL epitopes, we hypothesized certain naturally arising HLA- driven mutations may impair Nef›s ability to SERINC5 and affect patients› disease progression. Methods: Nef genes were PCR-amplified from plasma viral RNA of treatment- naïve, chronically HIV-1-infected subjects (N=375) recruited in Japan. The amplified fragments were directly sequenced and cloned in to a plasmid for functional assays of their gene products. Nef’s ability to counteract SERINC3/5 was assessed by infectivity of viral particles that were produced by co- transfection of NL43 and the patient-derived nef genes to Jurkat cells expressing SV40 Large T antigen (JTAg) and JTAg cells lacking expression of SERINC3/5. Results: The phylogenetically-informed statistical approach revealed that Y120F and Q125H mutations located close to the FPD 121-123 motif were significantly enriched in Nef sequences from subjects harboring HLA-B*51:01 that was known to present 120 YFPDWQNYT 128 as a CTL epitope. Interestingly, the number of the two mutations (120F/125H) was significantly inversely correlated with the plasm viral load; whereas no other HLA-associated polymorphisms

Icahn School of Medicine at Mt Sinai, New York, NY, USA Background: HIV-1 infection is associated with numerous comorbidities due to chronic inflammation. Purinergic (P2X) receptors are mediators of inflammatory signaling and have been increasingly implicated in HIV pathogenesis. Activation of P2X receptors can facilitate calcium influx, which then triggers downstream inflammatory signaling. Here we investigate whether HIV-1 binding or entry can activate P2X receptors and whether this receptor activation is associated with HIV-1 productive infection. Methods: MT4 cells loaded with Fluo-4 calcium-sensitive dye and onto the Beacon (Berkeley Lights) single-cell imaging platform. Cells were exposed to HIV-1 NL-CI, a fluorescent reporter virus that expresses mCherry in place of nef and nef is expressed on an IRES. Calcium influx was measured in short time intervals up to 20 minutes after exposure to virus by measuring the Fluo-4 (green) fluorescence. The same cells were tracked over 48 hours for development of mCherry signal to indicate HIV-1 productive infection. This experimental setup was repeated in parallel in the presence of NF449, a P2X receptor antagonist. Results: HIV-1 exposure was associated with an acute increase in intracellular calcium levels that corresponded to HIV-1 productive infection. Treatment with NF449 reduced HIV-stimulated calcium influx and HIV-1 productive infection. The higher magnitude of calcium influx was associated with higher levels of HIV-1 productive infection. Conclusion: The Beacon single cell imaging platform is a novel and effective tool for tracking both calcium influx and HIV-1 productive infection in cells. Our findings demonstrate that HIV-1 exposure can activate calcium influx through a mechanism that is sensitive to a P2X receptor antagonist. We observe that a P2X receptor antagonist reduces calcium influx and HIV-1 productive infection. These findings demonstrate that calcium signaling correlates with productive infection and indicates importance of calcium signaling in early infection events. Further development of P2X inhibitors as drugs could prove to be effective at both suppressing viral load and preventing inflammation- associated comorbidities. Background: The HIV-1 Nef accessory factor is essential for efficient viral replication and immune evasion in vivo. Nef homodimer formation affects interaction with host cell effectors, including the endocytic trafficking adaptor protein, AP-2. HIV-1 virions harboring dimerization-defective Nef show reduced infectivity and replication in cell culture. Here we explored the role of Nef dimerization and AP-2 recruitment in viral replication and CD4+ T cell loss in humanized mice. Methods: We generated mutants that are deficient for Nef dimerization (Ile 109/Leu 112/Tyr 115/Phe 121 to Asp; 4D mutant) and AP-2 binding (Asp 174/175 to Ala; DDAA mutant and Arg134 to Glu; RE mutant) based on previous X-ray crystal structures. A virus defective for Nef expression was also included (ΔNef mutant) as reference control. Effects of these mutations on viral infectivity and replication were investigated using TZM-bl reporter cells and CEM-GFP cells, respectively. BLT (bone marrow-liver-thymus) and hPBMC-NSG humanized mice were infected with each virus (2000 TCID50 equivalents/mouse), and replication measured by real-time quantitative RT-PCR targeting Gag or p24 AlphaLISA assays in plasma and tissues. Human CD4+ T cell counts were followed in peripheral blood and tissues by flow cytometry as a surrogate for HIV-1 pathogenesis. Results: In vitro, HIV-1 infectivity and replication were both significantly reduced with the 4D, DDAA, RE and ΔNef viruses. Humanized BLT mice infected with ΔNef viruses showed significantly lower viral loads and reduced CD4 depletion compared to wild-type HIV-1 within the course of 22 weeks post infection. hPBMC-NSG mice infected with the ΔNef and 4D mutant viruses showed decreased viral loads and displayed CD4+ T cell counts comparable to uninfected mice within the course of 6 weeks post infection. Nested PCR and nucleotide sequencing did not identify reversions of the 4D mutant recovered from humanized mice. However, a possible reversion was found in one viremic mouse infected with the RE mutant. Conclusion: Our results demonstrate that Nef homodimerization is important for HIV-1 pathogenesis in humanized mouse models of HIV/AIDS. These data Sherry Shu , Li Chen, Tom E. Smithgall University of Pittsburgh, Pittsburgh, PA, USA

Poster Abstracts

188 HOMODIMERIZATION IS REQUIRED FOR HIV-1 NEF FUNCTION IN HUMANIZED MICE

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CROI 2019