CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

157 HIV-1 RESISTANCE TO KF116 REVEALS INTEGRASE’S ROLE DURING POLYPROTEIN PROCESSING Ashley C. Hoyte 1 , Augusta V. Jamin 2 , Pratibha Chowdary Koneru 1 , Stephanie Rebensburg 1 , Jason Anderson 1 , Matthew Kobe 1 , Emma N. Ivanauskas 1 , James R. Fuchs 3 , Alan N. Engelman 2 , Mamuka Kvaratskhelia 1 1 The Ohio State Univ, Columbus, OH, USA, 2 Dana-Farber Cancer Inst, Boston, MA, USA, 3 The Ohio State Univ, Columbus, OH, USA Background: The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct sub-class of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing aberrant IN multimerization, while being ineffective during the early steps of viral replication. Here, we investigated the mechanism for evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture, with a representative MINI KF116. Methods: The mutant viruses were generated by introducing the substitutions in the IN coding sequence of HIV-1NL4-3 molecular clone using site-directed mutagenesis. Viral particles were prepared in HEK293T cells and transmission electron microscopy was utilized to visualize viral particle morphologies. In addition, viral protein processing was monitored using immunoblotting. The structure of the mutant catalytic core domain (CCD) dimer containing T124N/V165I/T174I IN substitutions was determined by X-ray crystallography. Surface plasmon resonance experiments were performed to determine K d values for KF116 binding to wild type and mutant CCDs. Results: We show that HIV-1NL4-3 IN(T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the CCD, yet at the same time, markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol and resulted in immature virions. Conclusion: Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, structural studies with CCD (T124N/ V165I/T174I) dimer compared to its wild type counterpart suggest a path for rationally developing second generation MINIs. 158 ROLE OF MA TRIMERIZATION IN HIV-1 ENVELOPE GLYCOPROTEIN INCORPORATION Mariia Novikova , Philip R. Tedbury, Eric O. Freed NCI, Frederick, MD, USA Background: The HIV-1 envelope (Env) glycoprotein complex traffics to the plasma membrane as a heterotrimer containing three molecules each of the surface glycoprotein gp120 and the transmembrane glycoprotein gp41. The gp41 subunit contains a long cytoplasmic tail that interacts with, or is accommodated by, the matrix (MA) domain of Gag during viral assembly. It has been proposed that hexamers of MA trimers form in membrane-bound Gag, overlaying the hexameric capsid lattice. Mutations at the tips of the putative MA trimers (which face the central aperture of the hexamer) prevent the incorporation of Env into particles. The goal of this study was to elucidate the role of MA trimerization in HIV-1 Env glycoprotein incorporation. Methods: We recently developed a glutaraldehyde cross-linking assay, using a virus with two lysine residues located near the MA trimer interface to analyze MA trimer formation in both immature and mature viral particles. This virus-based MA trimerization assay has enabled us to probe the role of MA trimer formation in Env incorporation. Results: MA residue L74 is critical for MA trimerization and Env incorporation. This residue is located in the hydrophobic region of the MA trimer interface. Two mutant viruses, L74E and L74G, do not incorporate Env and do not replicate in T cells. MA trimers were also not detected in these viruses. We have selected compensatory mutations that rescue the Env incorporation defect imposed by these mutations. Interestingly, two identified mutations, F43I and F43L, are located in the hydrophobic core of the trimer interface close to L74. We showed that both mutations F43I and F43L, in combination with another mutation, V34I, completely rescued Env incorporation and restored virus replication and infectivity. We are currently using a variety of approaches to monitor the formation of MA trimers in the replication-competent V34I/F43I/L74E and V34I/F43I/L74G mutants. Conclusion: We demonstrated that alterations in the structure of the MA trimer interface affect Env incorporation in assembled virions. Our findings suggest that MA trimer formation is a necessary step for Env incorporation during production of viral particles. These data may ultimately be useful for development of novel therapeutics targeting the MA trimer interface and Env incorporation. 159 PRODUCTION OF HIV-1 PROTEINS FROM “DEFECTIVE” HIV-1 PROVIRUS Hiromi Imamichi 1 , Joseph Adelsberger 2 , H. Clifford Lane 1 1 NIAID, Bethesda, MD, USA, 2 Leidos Biomed Rsr, Inc, Frederick, MD, USA Background: The presence of “defective” proviruses in HIV-infected patients has been well documented. As these defective proviruses are unable to encode intact viruses, they have been thought of as a silent graveyard of viral sequences. Contrary to this notion, we have recently reported that these “defective” proviruses transcribe novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy including those with HIV-1 RNA levels <50 copies/ml. In the present study, we demonstrate the emergence of these defective proviruses during in vitro infection and their association with HIV-1 protein production in the absence of intact virions. Methods: Single-cell clones were isolated from an H9 T-lymphoid cell line chronically infected with the MN strain of HIV-1. The H9 culture was first separated based on different cell surface characteristics of CD3 and HIV-1 Env gp120 expression by fluorescence-activated cell sorting. Positive wells by HIV-DNA PCR were further cloned by serial dilution at a cell density of 1 cell per well in 96-well plates. The identification of single-cell clones harboring “defective” proviral DNA was confirmed by combining 5’LTR-to-3’LTR single- genome amplification and direct amplicon sequencing of the genomic DNA. RNA transcription in the clones of interest was assessed by RT-PCR of near full-length unspliced HIV-1 RNA species. Cellular expression of HIV-1 proteins was analyzed by western blot and flow cytometry. Results: Twelve HIV-1 positive individual clones were isolated. Four harbored intact proviruses and expressed Gag p55/p24, RT p66/p51, integrase and Env gp160/gp120 proteins. The remaining eight clones had lethal +1 frameshift mutations in the reverse transcriptase (RT) gene (nucleotide position 3204 of the HXB2), resulting in premature termination of RT translation at Asp 218. Consistent with the DNA and RNA data, western blots revealed the presence of Gag p55/p24 and Env gp160/gp120 proteins and a complete absence of RT p66/p51 and integrase proteins in these eight clones. Conclusion: In the present study, we demonstrated asynchronous HIV-1 protein production from cells harboring “defective” proviruses that closely resembled the “defective” proviral species found in vivo in patients with HIV-1 infection. The proteins encoded by these “zombie” proviruses may serve as a cause of persistent immune activation during suppressive HIV treatment and may represent a significant hurdle to an HIV-1 cure. 160LB NUCLEAR PORE HETEROGENEITY AFFECTS HIV-1 INFECTION AND THE ANTIVIRAL ACTIVITY OF MX2 Melissa Kane 1 , Stephanie Rebensburg 2 , Matthew Takata 1 , Trinity Zang 1 , Masahiro Yamashita 3 , Mamuka Kvaratskhelia 2 , Paul Bieniasz 4 1 The Rockefeller Univ, New York, NY, USA, 2 The Ohio State Univ, Columbus, OH, USA, 3 Aaron Diamond AIDS Rsr Cntr, New York, NY, USA, 4 Howard Hughes Med Cntr, New York, NY, USA Background: Mx2 (myxovirus resistance 2) is an interferon-induced inhibitor of the preintegration phases of HIV-1 infection that is localized to the nuclear pore. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to Mx2 and mutations in the HIV-1 capsid protein that are known or suspected to alter the nuclear import pathways used by HIV-1 alter susceptibility to Mx2. Methods: We explored the complex interactions between Mx2, the viral CA, and the nuclear pore complex using a systematic knock-down approach with a panel of siRNAs targeting nucleoporins and nuclear import factors. Results: We found that cell-type, cell cycle, cyclophilin A, the viral CA, and multiple nucleoporins affect sensitivity to Mx2. Nucleoporin expression levels vary in cell lines commonly utilized in HIV-1 experimentation, and nucleoporin depletion affects HIV-1 infection in a cell-type and CA dependent manner. We observed that the subcellular localization of Mx2 is dependent upon nucleoporins, and that Mx2 is co-localized with some nucleoporins to a greater degree than others. We also pinpoint individual nucleoporins

Poster and Themed Discussion Abstracts

62

CROI 2017