CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts


165 HIV-1 CAPSID-NUCLEAR ENVELOPE INTERACTIONS THAT FACILITATE NUCLEAR IMPORT Mohamed Husen Munshi 1 , Ryan C. Burdick 1 , Wei-Shau Hu 1 , Vinay K. Pathak 1 1 National Cancer Institute, Frederick, MD, USA Background: HIV-1 must enter the nucleus and integrate its DNA into host genome for successful infection. However, the mechanism by which the viral complex docks at the nuclear envelope (NE) and enters the nucleus is not well understood. Although CA is known to play a critical role in nuclear import, the CA determinants that influence NE docking and viral complex translocation through the nuclear pore have not been defined. To identify the critical CA determinants, we developed quantitative live-cell imaging assays to study the NE docking and nuclear import of single viral complexes. Methods: A high-throughput live-cell imaging assay was developed to study NE docking and residence times of single viral complexes labeled with either HIV-1 integrase-superfolder green fluorescent protein (sfGFP), APOBEC3F- yellow fluorescent protein (A3F-YFP) or Cyclophilin A-red fluorescent protein (CypA-DsRed). The amount of CA associated with viral complexes was quantified using a newly developed direct CA label (GFP-CA); CA was also detected by immunostaining with anti-CA antibody. Infectivity was determined in HeLa cells, and the CEM-SS and MT4 T cell lines. Results: Using high-throughput live-cell imaging, we identified CA mutants M10I, M10V and I15V that exhibited longer NE residence times compared to wild-type viral complexes in a CypA-dependent manner. Additionally, the M10 mutant complexes that entered the nucleus had longer NE residence times compared to WT, but only for the CypA-dependent nuclear import pathway. Analysis of virions labeled with CypA-DsRed also indicated that most viral complexes lost CypA-DsRed prior to nuclear import. CA mutants did not show infectivity defects in HeLa cells but were defective in T cell lines. Direct labeling of CA (GFP-CA) indicated that the CA levels of WT and mutant viral complexes were similar; however, CA detection using anti-CA antibody suggested differences in mutant viral complexes that reduced anti-CA antibody binding as a result of differences in conformation or host protein binding. Conclusion: We have identified CA determinants that play a critical role in NE docking and nuclear import in a CypA-dependent manner. We propose a model in which CypA stabilizes the initial interaction of the viral core with NE but does not enter the nucleus with the viral core. These studies provide valuable insights into the interactions between the viral complex and the NE that result in stable docking and nuclear import. 166 REPORTER VIRUSES WITH PROTEIN BARCODES TO ANALYZE HIV LATENCY ESTABLISHMENT Eun Hye G. Kim 1 , Maria C. Bermúdez-González 1 , Michael Schotsaert 1 , Ana Fernández-Sesma 1 , Lubbertus C. Mulder 1 , Lara Manganaro 2 , Viviana A. Simon 1 1 Icahn School of Medicine at Mt Sinai, New York, NY, USA, 2Istituto Nazionale Genetica Molecolare, Milan, Italy Background: Studies of HIV latency establishment at the single cell level have been hampered by difficulties to identify CD4+ T cells that harbor transcriptionally silent proviruses. HIV molecular clones encoding reporter proteins, whose expression is either dependent or independent of HIV LTR promoter, have been powerful tools to dissect mechanisms of HIV persistence. To better support multi-dimensional analyses such as those carried out by Mass Cytometry (CyTOF), we have generated HIV dual reporter viruses that carry cell membrane expressed protein barcodes. Methods: To detect the LTR independent expression of affinity tags such as chimeric protein V5-NGFR or the GFP reporter protein, a PGK promoter driven reporter cassette was cloned into the envelope frame of HIV molecular clone pLAI2. In addition, for the assessment LTR-dependent expression, reporters HSA-mCherry were cloned upstream of an internal ribosomal site followed by nef. V5-NGFR and HSA, in contrast to GFP or mCherry, are both expressed at the plasma membrane of the cell making the reporters easily accessible to membrane probes and magnetic bead enrichment approaches.

Results: Primary human CD4+ T cells from six different donors were stimulated with IL-2, IL-15 or CD3/CD28 and infected with the different dual reporter viruses. Cells were analyzed by flow cytometry after 3, 4 and 5 days to determine the optimal conditions and select the most informative time points and donors. The reporter virus HIV-GKO previously described by the Verdin lab was used as reference. Mock infected and HIV infected cells were analyzed by CyTOF. We used a customized CyTOF antibody panel, which captures 30 different markers allowing the discrimination of five different CD4+ T populations including CD4+ T memory cells with stem cell like properties (CD4+ Tscm) and CD4+ Tregs. Markers for determining cellular features such as proliferation, activation and cell cycle were also included. CyTOF experiments were performed at the Human Immune Monitoring Center of the Icahn School of Medicine. Conclusion: Our preliminary data indicate that our dual reporter viruses allow accurate detection of both LTR silent and LTR active proviruses with minimal promoter interference. We will expand on the existing viruses to generate panels of barcoded reporter viruses to test the influence that viral genes, such as integrase and Vpr, have on latency establishment and maintenance in specific primary human CD4+ T cell populations. 167 Gag DETERMINANTS OF SPECIFIC GENOME PACKAGING IN HIV-1 AND HIV-2 Jonathan Rawson 1 , Olga A. Nikolaitchik 1 , Xayathed Somoulay 1 , Jennifer A. Yoo 1 , Vinay K. Pathak 1 , Wei-Shau Hu 1 1 National Cancer Institute, Frederick, MD, USA Background: HIV packages a dimer of its RNA genome into virus particles. The viral protein Gag drives this process by binding to the packaging signal in the 5’ untranslated region of genomic RNA. However, Gag also binds cellular RNAs, and the mechanism by which Gag selectively packages the viral genome remains poorly understood. It was previously observed that HIV-1 and HIV-2 exhibit a striking difference: HIV-1 Gag efficiently packages HIV-2 RNA, but HIV-2 Gag does not package HIV-1 RNA. We hypothesized that studies of these non- reciprocal interactions would lead to the identification of novel HIV packaging determinants. Methods: HIV-1-based Gag chimeras were constructed that contained the entire HIV-2 nucleocapsid (NC) domain or just the two zinc fingers of HIV-2 NC. The chimeras were transfected into 293T cells, and Gag expression, particle release, and maturation were examined. Single virion analysis, a technique in which individual particles are analyzed by fluorescence microscopy, was performed to determine packaging efficiencies for HIV-1 or HIV-2 RNA. Spreading infections were conducted in MT-4 T cells to determine replication competence and to select for adaptive mutations. Results: The chimeras did not affect Gag expression or particle release but did slightly impair Gag processing. Surprisingly, both chimeras packaged HIV-1 RNA into ~70% of particles, a modest reduction relative to wild-type (WT) HIV-1 (~95%). However, when HIV-1 and HIV-2 RNAs were co-expressed and competed for packaging, both chimeras strongly preferred to package HIV-2 RNA. In contrast, WT HIV-1 Gag packaged HIV-1 and HIV-2 RNAs with similar efficiencies. We further found that the chimeras replicated in MT-4 cells, although with delayed kinetics compared to WT HIV-1. When re-passaged, the chimeras replicated significantly faster, indicative of adaptation. Putative adaptive mutations in Gag were identified by PCR and sequencing. One single amino acid substitution was found in the first zinc finger of HIV-2 NC and represents a switch from an HIV-2 to an HIV-1 residue at this position. This mutation alone significantly improved chimera replication. Conclusion: Our findings provide new insights into the mechanistic basis of selective genome packaging in HIV-1 and HIV-2. These studies may inform future efforts to develop antivirals targeting RNA packaging and have implications for the possible emergence of HIV-1/HIV-2 recombinants in co- infected individuals.

Poster Abstracts


CROI 2020

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