CROI 2019 Abstract eBook

Abstract eBook

Oral Abstracts

of these are essential for virus replication. Here, we report a proteomics-to- genetics approach to assess the functional roles of HIV-human protein-protein interactions in primary CD4+ T cells. Methods: Leveraging a high-throughput CRISPR-Cas9 platform for primary T cell genome engineering, we targeted 435 host factors previously identified to physically interact with HIV proteins for knock-out in CD4+ T cells frommultiple donors. Each population was subject to deep sequencing to quantify editing efficiency and concurrently challenged with replication-competent HIV-1 to assess the impact on HIV infection. Results: Using this platform, we achieved robust editing efficiencies with high donor-to-donor concordance, averaging 75% allelic knock-out at the population level. The repair outcomes at each edited site demonstrated remarkable predictability based on the target site sequence and surrounding chromatin structure. Of the 435 targeted genes, we identified 86 HIV host factors, 47 of which have not been previously reported. While most host factors were conserved between donors, several displayed notable donor variation. These factors were temporally separated into early and late-acting genes and physically segregated by HIV interacting protein, greatly facilitating and expediting functional analyses. Mechanistic interrogation revealed critical roles for these new HIV host factors in viral entry, transcription, budding, and maturation. Conclusion: These findings reveal several new host factors underlying HIV replication in primary CD4+ T cells and model an interdisciplinary approach to systems biology as a means to streamline experimental discovery. Donor-to- donor and cell type-to-cell type variations in host factor dependency suggest the virus employs substantial functional plasticity to achieve robust infection, complicating host-based therapeutic strategies. The continued extension of this technology to resting memory T cells and for the targeted insertion of single nucleotide variants will ultimately unveil new insight into the host determinants underlying HIV replication, latency, and pathogenesis.

together on microtubules. However, a mutation in capsid that abolishes binding to CPSF6, N74D, rendered the virus unable to associate with cytoplasmic CPSF6. Disruption of microtubule polymerization resulted in diminished virus and CPSF6 movement. Truncation or mutation of the RS domain of CPSF6 led to reduced binding to TNPO3 and increased cytoplasmic expression at the cell periphery, resulting in restriction of HIV-1 infection. This CPSF6 mislocalization resulted in the formation of higher-order complexes around HIV-1 IN-containing complexes, premature capsid uncoating, and altered microtubule trafficking of IN complexes after infection with WT HIV-1 but not N74D HIV-1. In addition, CRISPR-mediated knockout of the CPSF6 gene in cells altered microtubule- mediated trafficking towards the nucleus of WT HIV-1 but not the capsid mutant. Conclusion: These data suggest that after WT HIV-1 entry into the cell, viral complexes interact with CPSF6 and TNPO3 on microtubules near the nucleus, which is required for efficient capsid uncoating and nuclear entry of pre- integration complexes. 72 HIV-1 CAPSID DETERMINANTS THAT INFLUENCE NUCLEAR ENVELOPE DOCKING AND NUCLEAR IMPORT Mohamed Husen Munshi , Ryan C. Burdick, Wei-Shau Hu, Vinay K. Pathak National Cancer Institute, Frederick, MD, USA Background: An essential step of HIV-1 infection is to transfer the replication complex into the nucleus. An HIV-1 intact viral core is approximately 61-nm wide must get translocated through 39-nm-diameter nuclear pores, suggesting that the viral core undergoes uncoating and/or conformational changes before entering the nucleus. While HIV-1 capsid (CA) protein plays a critical role in nuclear import, the CA determinants that influence nuclear envelope (NE) docking and viral complex translocation through the nuclear pore have not been defined. To study these events, we developed a quantitative imaging assay for association of single viral complexes with the NE and for their nuclear import. Using this system, we evaluated several CA mutants in which core surface- associated amino acids were substituted and determined their ability to dock at the NE and/or enter the nucleus. Methods: HIV-1 CA mutants, including hyperstable (E45A) and hypostable (P38A) mutants, were generated in envelope-deficient genomes. VSV-G pseudotyped virions were produced and used to determine their infectivity in HeLa, CEM-SS, and MT4 cells. For imaging assays, HIV-1 virions were labeled with HIV-1 integrase-superfolder green fluorescent protein (sfGFP) and used to study NE docking and nuclear import in both fixed-cell and live-cell assays. A high-throughput live-cell imaging assay was developed to study NE-docking and residence time of CA mutants. HIV-1 CA amounts were determined using a quantitative immunostaining assay. Results: We identified CA mutants that exhibited a longer NE residence time compared to wild-type viral complexes, indicating that these CA determinants can influence the kinetics of association of viral complexes with the nuclear pore. These CA mutants did not show infectivity defects in Hela cells, but were defective in T cell lines (CEM-SS and MT4 cells). Interestingly, viral complexes of these mutants docked at the NE exhibited lower CA signals in immunofluorescence assays, suggesting alterations in the viral core structures. Live-cell imaging experiments are being performed to determine whether the CA mutants increased the NE residence time of those viral complexes that enter the nucleus. Conclusion: We have identified CA mutants that exhibit long NE residence times, indicating defects in NE association, a phenotype which has not been previously reported. Further characterization of these CA mutants may provide valuable insights into the essential steps of NE docking. 73 SINGLE HIV-1 VIRUS IMAGING WITH CA-EGFP QUESTIONS A ROLE OF NUCLEAR CA IN INTEGRATION Irena Zurnic, Lieve Dirix, Veerle Lemmens, Doortje Borrenberghs, Susana Rocha, Johan Hofkens, Frauke Christ , Jelle Hendrix, Zeger Debyser Katholieke University Leuven, Leuven, Belgium Background: The role of HIV-1 capsid protein (CA) in early HIV replication is known to extend beyond uncoating. Still, a consensus model on the relationship between HIV-1 uncoating, nuclear import and integration is lacking, mostly due to conflicting results on intracellular capsid distribution. Resolving the dynamics of capsid uncoating thus necessitates a robust method of imaging functional viruses containing labeled CA.

Oral Abstracts

71 HIV-1 COMPLEXES TRAFFIC WITH HOST CPSF6 ON MICROTUBULES PRIOR TO NUCLEAR ENTRY Zhou Zhong 1 , Douglas K. Fischer 1 , Chris Kline 1 , Sooin Jang 2 , Alan N. Engelman 2 , Simon C. Watkins 1 , Zandrea Ambrose 1 1 University of Pittsburgh, Pittsburgh, PA, USA, 2 Dana–Farber Cancer Institute, Boston, MA, USA Background: HIV-1 DNA nuclear entry is required for infection and is mediated by viral capsid. The host protein CPSF6 has been shown to bind HIV-1 capsid, to facilitate nuclear import of viral pre-integration complexes, and to mediate integration of viral DNA into actively transcribed genes. While CPSF6 has predominantly nuclear expression as a result of binding to host transportin TNPO3 via its RS domain, a small fraction of CPSF6 is localized outside of the nucleus, leading us to study its interaction with HIV-1 in the cytoplasm. Methods: In this study, we conducted high speed live-cell confocal imaging to investigate intracellular trafficking of WT or mutant HIV-1 containing functional, fluorescently tagged integrase (IN). Infection was performed in cells with fluorescently labeled microtubules, TNPO3, and full-length or mutant CPSF6. In addition, HIV-1 capsid uncoating kinetics were measured in infected cells using an imaging-based assay. Results: CPSF6 was expressed as puncta in the perinuclear region of the cytoplasm, which trafficked on microtubules with TNPO3. Upon infection, WT HIV-1 complexes associated with perinuclear CPSF6 and TNPO3, trafficking


CROI 2019

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