CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

168 EXPLORING THE REQUIREMENTS FOR HIV-1 GENOME PACKAGING THROUGH A NONVIRAL RNA BINDING Alice Duchon 1 , Steven Santos 1 , Jianbo Chen 1 , Vinay K. Pathak 1 , Wei-Shau Hu 1 1 National Cancer Institute, Frederick, MD, USA Background: HIV-1 efficiently selects and packages its RNA genome into assembling virus particles at the plasma membrane. The main viral structural component, Gag, orchestrates the complex interactions that occur during this stage. In the cytoplasm, Gag shows only a slight preference for binding HIV-1 RNA over cellular mRNA. Therefore, we hypothesized that in addition to RNA binding, Gag must use other mechanisms to ensure selective HIV-1 RNA genome packaging. To better define the mechanism of HIV-1 RNA packaging specificity, we designed an experimental system to study Gag:RNA interactions. Methods: To separate the specific and nonspecific Gag:RNA interactions, we created morphologically normal, empty, virus-like particles by replacing the NC domain of Gag with a leucine zipper (LZ). To package RNA, we fused a bacterial RNA binding protein, BglG, to an internally mCherry-labeled GagLZ (GagLZiC- Bgl). BglG specifically binds to a stem loop RNA structure, BSL. Gag-expressing constructs contained two sets of stems-loops: BSL and sequences recognized by bacterial phage PP7 coat protein. Viral RNA was detected by coexpressed YFP fused to PP7 coat protein (PP7-YFP). To examine whether GagLZ, GagLZ-CFP fusion protein, and GagLZiC-Bgl proteins coassemble to generate viral particles with RNA, the constructs were cotransfected with PP7-YFP into human 293T cells. Harvested viral particles were imaged using fluorescence microscopy. Results: Most particles were CFP+ and iC+; thus, these Gag proteins coassembled into the same virus. Additionally, ~40% of the particles were also YFP+, indicating they contained viral RNA. To determine trans-acting requirements for RNA packaging, we created a series of GagLZiC-Bgl truncation mutants. RNA was efficiently packaged by mutants lacking the LZ motif or p6 domain. However, RNA packaging was significantly reduced when regions important for Gag oligomerization were deleted or when GagLZiC- Bgl myristylation was inhibited by a G1A mutation. These results reveal the importance of the Gag:RNA and Gag:Gag interactions at the plasma membrane for genome packaging. All mutant Gag constructs coassembled with GagLZ-CFP. Conclusion: Overall, we developed an experimental assay to identify properties of Gag required for RNA packaging during HIV-1 assembly. In an NC-independent system as well as in the presence of NC, the multimerization of RNA-bound Gag is needed for efficient RNA packaging. Haitao Guo 1 , Qi Wang 1 , Li Wang 1 , Khader Ghneim 2 , Elena Rampanelli 1 , Carolina Garrido 1 , Rafick-Pierre Sekaly 2 , Merlin L. Robb 3 , Leigh Anne Eller 3 , David M. Margolis 1 , Xian Chen 1 , Lishan Su 1 , Jenny P. Ting 1 1 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 2 Case Western Reserve University, Cleveland, OH, USA, 3Walter Reed Army Institute of Research, Silver Spring, MD, USA Background: Human Immunodeficiency Virus type-1 (HIV-1)-infected individuals showmetabolic alterations of CD4 T cells through unclear mechanisms. The nucleotide-binding, leucine-rich-repeat-containing protein NLRX1 is a novel host factor required for HIV-1 infection. Whether NLRX1 has an interaction with the immunometabolism to promote HIV-1 infection of CD4 T cells is an intriguing question. Methods: First, we silenced NLRX1 expression in human primary CD4 T cells by short hairpin RNA and assessed HIV-1 replication in those cells compared with the control cells. Second, we used quantitative mass spectrometry to profile the altered protein expression resulted from HIV-1 infection of Jurkat T cells, followed by an analysis of differentially expressed proteins between NLRX1-silenced cells and the control cells. Third, we conducted metabolic assays to compare the differentially induced oxidative phosphorylation (OXPHOS) and glycolysis by HIV-1 infection in NLRX1-silenced T cells vs the control cells. Fourth, we used the inhibitor and activator of OXPHOS to modulate HIV-1 replication in both primary CD4 T cell culture and human CD4 T cell-reconstituted mouse model. Finally, we analyzed the RV217 transcriptomic study of HIV-1 patients to search the association between immunometabolic pathways and HIV-1 viremia. Results: LRX1 facilitates HIV-1 replication in both human primary CD4 T cells and human CD4 T cells-reconstituted mice Quantitative proteomics and metabolic analyses reveal that NLRX1 enhances OXPHOS and glycolysis during HIV-1-infection of CD4 T cells to promote viral replication. Inhibition of OXPHOS by an FDA-approved drug, metformin, suppresses HIV-1 replication in primary 169LB MULTI-OMICS ANALYSES REVEAL IMMUNOMETABOLIC REPROGRAMMING-DEPENDENT HIV-1 REPLICATION

CD4 T cells and in humanized mice. Potentiating OXPHOS by resveratrol restored the deficiency of HIV-1 replication in NLRX1-silenced T cells. The role of OXPHOS during HIV-I infection in patients is supported by the transcriptome profiling of CD4 T cells from 22 male and 15 female HIV-1 patients residing in Asia and Africa. HIV-1 viremia positively correlates with NLRX1 expression and poor outcomes are associated with elevated OXPHOS. Conclusion: NLRX1 promotes HIV-1 replication in CD4 T cells by inducing immunometabolism OXPHOS and glycolysis. Inhibition of OXPHOS by metformin suppressed HIV-1 replication in both primary human CD4 T cells and humanized mice. OXPHOS is positively correlated with HIV-1 viremia in HIV-1 patients. This study uncovers a T cell OXPHOS pathway as an unappreciated target for HIV-1 therapy. Nathaniel L. Talledge 1 , Huixin M. Yang 1 , Luiza Mendonca 1 , Ke Shi 1 , Hideki M. Aihara 1 , Wei Zhang 1 , Louis M. Mansky 1 1 University of Minnesota, Minneapolis, MN, USA Background: Immature retroviruses like human immunodeficiency virus type 1 (HIV-1) are known to possess an unusual degree of irregularity, with Gag proteins forming a hexagonal lattice that drives assembly and release of particles lacking icosahedral symmetry, and creating a lattice this is substantially incomplete. Immature virus particle structure can provide important clues to the nature of virus particle assembly in cells. Methods: In this study, we sought to decipher key structural details of immature retroviral morphology of HIV-1 and the less pathogenic HIV-2 by obtaining high-resolution structures using cryo-electron microscopy (cryo- EM). In particular, we sought to identify a structural basis for our preliminary observations indicating that distinct differences in immature particle morphology are observed between HIV-1 and the less pathogenic HIV-2. In particular, key phenotypic features that distinguish HIV-2 immature particles include a larger average particle size as well as a nearly complete Gag lattice. Results: Structural comparison at 5.5 Å resolution between published HIV-1 and that of our HIV-2 cryo-EM reconstructions emphasizes the importance of the capsid (CA) C-terminal domain (CTD) and spacer peptide 1 (SP1) regions in forming hexameric assemblies of CA in the intermolecular contacts of the overall lattice structure (including critical residues at the dimeric and trimeric intermolecular interfaces). In conjunction to the cryo-EM analyses, we solved a 1.98Å crystal structure of HIV-2 CACTD and found a unique extra alpha helix (H12) at the C-terminal region, which was not previously observed in the domain structures of HIV-1 or other retrovirus CA proteins. Fitting of the HIV-2 CACTD into the reconstruction map confirmed critical contact interfaces of Gag proteins, and emphasized the importance of the co-factor inositol hexakisphosphate (IP6) at the six-fold symmetry interface. The presence of H12 in CA may contribute the stability of the hexametric interactions in the HIV-2 immature Gag lattice. Conclusion: Taken together, our observations provide important clues for explaining the observed morphological differences between immature HIV-1 and HIV-2 particles. These differences may help explain differences virus virulence. 171 B CELLS DIRECT R5-TROPIC HIV INFECTION OF CCR5 NEG NAIVE CD4+ T CELLS Abigail D. Gerberick 1 , Nicolas Sluis-Cremer 1 , Paolo A. Piazza 1 , Charles Rinaldo 1 , Giovanna Rappocciolo 1 , Diana Campbell Delucia 1 1 University of Pittsburgh, Pittsburgh, PA, USA Background: Naive CD4 T (TN) cells are an important reservoir of latent, replication-competent HIV. CD4 TN isolated from peripheral blood are resistant to direct infection with R5-tropic HIV in vitro because there is negligible expression of CCR5 on the cell surface. Paradoxically, R5-tropic virus has been isolated from TN cells from HIV-infected individuals on antiretroviral therapy. We assessed whether antigen presenting cells (APCs) - B cells and dendritic cells (DCs) - mediate trans infection of R5-tropic HIV to TN cells in the absence of global T cell activation. Methods: Total CD4 T cells, CD4 TN cells, B cells and monocytes were purified from PBMCs of seronegative donors by magnetic microbead separation. B cells were activated by CD40L and IL4, and DCs were differentiated frommonocytes by GM-CSF and IL4. B cells and DCs were pulsed with 10-3 moi R5-tropic HIVBaL and cultured with TN or total CD4+ T cells at a 1:10 ratio. As a control, we exposed TN or total CD4 T cells to 10-1 moi of HIVBaL in the absence of B cells or 170LB HIGH-RESOLUTION PARTICLE STRUCTURE OF IMMATURE HIV-2

Poster Abstracts

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CROI 2020