CROI 2017 Abstract e-Book

Abstract eBook

Oral Abstracts

Results: In addition to previously known NPC co-factors, we identify Nup35 and POM121 to aid HIV-1 infection. HIV-1 reliance on Nup35 or POM121 is linked to CA. Nup35 and POM121 are FG-Nups. Preliminary examination indicates that a C-terminal FG-motif of Nup35 is required for HIV-1 infection. Notably, HIV-1 interaction with CypA regulated dependence on the FG-Nups. Disruption of the interaction between CA and CypA either by cyclosporine A (CsA) treatment or CypA knockdown restored WT HIV-1 infectivity in Nup35 knockdown cells. Conclusion: We hypothesize that CypA use by HIV-1 prevents access to the N74 pocket in CA until the virus docks at the NPC. We propose that the HIV-1 core, comprised of hundreds of CA molecules, directly functions as a NTR and exploits successive FG interactions to achieve nuclear entry. Thus, the development of pharmacological compounds targeting the CPSF6-binding site in CA have the potential to disrupt interactions with multiple co-factors and significantly impair HIV-1 infection. 19 COMPREHENSIVE CRISPR SCREEN IDENTIFIES NOVEL HIV RESTRICTION FACTORS Molly Ohainle , Louisa Pendergast, Jolien Vermeire, Michael Emerman Fred Hutchinson Cancer Rsr Cntr, Seattle, WA, USA Background: Interferon-Stimulated Genes (ISGs) inhibit HIV replication by inducing an array of antiviral effectors known as HIV restriction factors. Restriction factors, such as TRIM5, APOBEC3s, Tetherin and MxB, have previously been discovered one-by-one through classic techniques such as cDNA library screening and comparison of RNAseq data in permissive versus non-permissive cells. Here we describe a comprehensive novel CRISPR gene knockout screen to identify HIV restriction factors. Methods: A CRISPR knockout screen was performed for wild type HIV-1 in THP-1 cells, a human monocytic cell line with strong Interferon-induced inhibition of HIV-1 replication. An available whole-genome CRISPR library was modified to allow for packaging of lentiCRISPR genomes in trans into budding HIV-1 particles after infection. Known and novel, candidate restriction factors are identified by measuring the relative enrichment of gRNAs in HIV-1 viruses released from cells (MAGeCK gRNA and gene analyses). Further, we have designed an ISG-specific gRNA CRISPR library targeting ~2000 human ISGs allowing for validation of hits from the whole-genome screen as well as identification of additional restriction factor candidate genes. Results: Our screen identified known restriction factors, such as MxB, ZAP and ISG15. Potential previously-uncharacterized HIV restriction factors were also identified such as Nedd4-binding protein 1 (N4BP1), a nucleolar protein previously identified as an ISG that participates in PML bodies and is an inhibitor of the ubiquitin ligase ITCH. Conclusion: We have developed a novel method to uncover HIV restriction factors. Both known and novel HIV restriction factors were identified. Our studies show that a subset of these genes can explain most of the inhibitory effects of interferon on HIV replication in THP-1 cells. Background: Most enveloped viruses, including HIV-1, exploit the endosomal sorting complexes required for transport (ESCRT) pathway to bud from cells. Owing to its essential functions in membrane fission events such as cytokinetic abscission and closure of the post-mitotic nuclear envelope, the ESCRT machinery is conserved and evolutionarily constrained. These properties pose a challenge for cells in trying to adapt to pathogens that exploit the ESCRT pathway. Methods: Using phylogenetic analyses, we identified retrotransposed copies of numerous ESCRT factors in primate genomes including a retrogene encoding a truncated version of the ESCRT-III protein CHMP3 (retroCHMP3) in squirrel monkeys. Truncated CHMP3 proteins had previously been shown to block HIV-1 budding, apparently by dominantly inhibiting the ESCRT pathway (Zamborlini et al., 2006). We therefore hypothesized that retroCHMP3 might also inhibit the budding of HIV-1 and other enveloped viruses, and tested this hypothesis by expressing retroCHMP3 in human cells and measuring inhibition of retrovirus budding and infectivity. Evolutionary reconstruction and functional analysis of chimeric proteins showed crucial differences between retroCHMP3 and the parental CHMP3 protein. Results: We found that both squirrel monkey retroCHMP3 and the analogously truncated endogenous CHMP3 protein potently inhibit budding of HIV-1 and other retroviruses. Importantly, however, retroCHMP3 exhibited much less cellular toxicity than the truncated parental protein. Characterization of chimeric constructs revealed that just seven amino acid changes were largely responsible for retroCHMP3 detoxification. Cytotoxicity correlated with the formation of cytoplasmic CHMP3 punctae, suggesting that the truncated parental CHMP3 protein may block both viral and cellular ESCRT pathways by sequestering essential ESCRT factors into insoluble aggregates, whereas retroCHMP3 has evolved to maintain viral inhibition without sequestering essential ESCRT factors. Conclusion: Our studies identify retroCHMP3 proteins as broad-spectrum inhibitors of enveloped virus budding in NewWorld monkeys. Moreover, retroCHMP3 has retained the ability to inhibit viral budding while apparently evolving to lose cellular cytotoxicity, revealing unexpected separation of cellular and viral ESCRT functions. More generally, our work illustrates how retrotransposition can create opportunities for cells to evolve new antiviral activities and counteract pathogen exploitation of essential host pathways. Background: The HIV-1 Gag polyprotein contains membrane binding, assembly and budding activities that are required to create extracellular virions that can transfer the viral genome from infected producer cells to new, uninfected target cells. These activities also underlie the efficacy of retroviral vectors, which have been explored extensively as potential therapeutic delivery vehicles. However, safety concerns, immunogenicity and inefficient packaging of non-nucleic acid cargoes limit their potential use. To overcome some of these limitations and to test our understanding of the fundamental principles of virion assembly and release, we have undertaken the design of new proteins that can self-assemble into enveloped protein nanocages (EPNs) and induce their own release from human cells. Methods: We characterized: 1) cellular release of EPNs using western blot assays of cell culture supernatants, 2) membrane integrity of released EPNs using antibody and protease susceptibility, 3) EPN vesicle architectures using cryo-EM tomography, 4) nanocage structures using high resolution single particle cryo-EM reconstructions, and 5) target cell fusion and enzymatic cargo delivery by VSV-G pseudotyped EPNs using colorimetric assays of packaged β-lactamase-Vpr fusion proteins. Results: We observed robust EPN assembly and release, which required all three design elements: membrane binding, self-assembly, and ESCRT factor recruitment. The overall strategy was very general and we have identified 16 different combinations of membrane binding, assembly and ESCRT recruiting elements that can produce EPNs. Detailed analyses of one EPN design (termed EPN-01) revealed that the protein nanocages assembled precisely as designed, and were released within membrane vesicles (110 nm average diameter), each of which contained multiple nanocages (14 nanocages/vesicle average). Pseudotyping with VSV-G allowed the EPNs to fuse with new target cells and deliver β-lactamase-Vpr cargoes. Conclusion: We have used enveloped virus assembly principles to design new proteins that can induce the formation extracellular vesicles and transfer their contents between cells. 20 A RETROTRANSPOSED ESCRT-III FACTOR BLOCKS HIV-1 BUDDING WITHOUT INDUCING CYTOTOXICITY Lara Rheinemann , Diane Downhour, Gaelle Mercenne, Alesia McKeown, John McCullough, Wes Sundquist, Nels Elde Univ of Utah, Salt Lake City, UT, USA 21 DESIGNED PROTEINS INDUCE THE FORMATION OF NANOCAGE-CONTAINING EXTRACELLULAR VESICLES Joerg Votteler 1 , Cassie Ogohara 2 , Sue Yi 2 , Yang Hsia 2 , Una Nattermann 2 , David M Belnap 1 , Neil P King 2 , Wes Sundquist 1 1 Univ of Utah, Salt Lake City, UT, USA, 2 Univ of Washington, Seattle, WA, USA

Oral Abstracts

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CROI 2017