CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

Conclusions: We describe a High Content imaging approach to identify novel host targets of HIV-1 VPU. Our results indicate that VPU targets multiple putative restriction factors for degradation. Elucidating the role of identified hits in counteracting HIV-1 infection would greatly enhance our understanding of VPU dependent enhancement of HIV-1 infection. Comprehensive understanding of virus dependent modulation of host immune machinery would significantly assist in developing effective anti-viral measures. 41 HIV-1 Adaptation to Humans Involved Interactions of Vpr With the DNA Damage Response Oliver I. Fregoso ; Michael Emerman Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA, US Background: The accessory protein Vpr is encoded by all extant primate lentiviruses and is important for viral replication in vivo . HIV-1 Vpr interacts with the host Cul4-DCAF1 ubiquitin ligase complex as well as the SLX4 complex of structure-specific nucleases, which have been implicated in the innate immune response. In addition, Vpr expression leads to G2 cell cycle arrest and activation of the DNA damage response after infection. We asked whether or not these functions of Vpr are related and what role adaptation of HIV-1 to humans played in shaping these interactions. Methods: We cloned Vpr from a diverse array of primate lentiviruses. Through evolutionary analyses, we generated a phylogenetic framework of these Vpr isolates and tested them for their ability to interact with the human Cul4-DCAF1 complex and the human SLX4 complex by co-immunoprecipitation. We assayed for the ability of Vpr orthologs to activate the DNA damage response by immunofluorescence for DNA damage-associated foci and by cell-cycle analyses. Results: We have identified that all Vpr orthologs are able to interact with the human Cul4-DCAF1 complex. Furthermore, we have found that a subset of these Vpr proteins can activate the DNA damage response in human cells. However, DNA damage induced by Vpr does not directly correlate with ability of Vpr to recruit the SLX4 complex. Instead, this function arose in select lentiviral lineages, including HIV-1 but not HIV-2. Conclusions: The recruitment of Cul4-DCAF1 is a conserved ancestral function of Vpr and indicates that all Vpr orthologs have the potential to activate the DNA damage response in their hosts. As activation of the DNA damage response and SLX4 complex recruitment do not correlate, this indicates that Vpr activates host DNA damage response pathways through a still unknown mechanism. Moreover, our data suggests that recruitment of this complex occurred after the cross-species transmission of lentiviruses from chimpanzees to humans, and is therefore important for the adaptation of HIV-1 group M to humans. 42 The HIV-1 Protease Can Interact With RNA to Dramatically Enhance Its Activity Marc Potempa 1 ; Ellen Nalivaika 2 ; Sook-Kyung Lee 1 ; Celia A. Schiffer 2 ; Ronald Swanstrom 1 1 University of North Carolina, Chapel Hill, NC, US; 2 University of Massachusetts Medical School, Worcester, MA, US Background: In the maturation step of the HIV-1 lifecycle, the virus-encoded protease (PR) must process the structural polyprotein Gag to generate infectious virus particles. Previous reports found that cleavage of the p15NC maturation intermediate accelerates when RNA is present. Our studies have shown RNA-enhancement is substrate-independent. We hypothesize that enhancement results from an interaction between PR and RNA. Methods: In vitro proteolysis reactions were performed +/- RNA with globular substrates derived from Gag or a 12-amino acid peptide. HIV PRs of multiple lineages were used to investigate whether RNA-enhancement is general to HIV. Alternative nucleic acids were also employed. Gel-shift assays were used to examine interactions between proteins and nucleic acids. Results: Addition of RNA to two-substrate in vitro proteolysis reactions containing Gag derivatives p15NC and MA/CA accelerated cleavage of both substrates. In reactions with only MA/CA, the rate increased 80x (see figure). Cleavage of a peptide substrate, too small to interact with RNA and be simultaneously cleaved, was accelerated 20x. These data argue that RNA enhancement is substrate-independent. RNA enhanced the activity of Subtype B, C, and AE PRs; however, the HIV-2 PR was unaffected. Select short DNA oligonucleotides (~20 bases) capable of forming homomultimers could increase PR activity, but to a lesser degree than long, heteropolymeric RNA. The PR preferentially interacted with the multimeric forms of oligos in gel-shift assays, implicating the DNA complexes as necessary. Dimer stability is unaffected by RNA, since the tethered-dimer was equivalent to the wild-type PR in our assays. RNA lowered the K m by 5-10x, and raised the V max 2-5x.

Oral Abstracts

Conclusions: RNA can directly interact with HIV-1 PRs in vitro , dramatically increasing their catalytic activity. This interaction likely relies on electrostatic forces, rather than sequence, since multimer formation was the principle determinant of effectiveness for DNA oligonucleotides. Mechanistically, RNA likely affects PR function post-dimerization, since RNA equivalently enhanced the wild type and tethered-dimer PRs. The differences in K m and V max show that RNA increases the PR’s affinity for its substrate and its turnover capacity. These data raise the possibility that PR cleavage during assembly is regulated in part by the juxtaposition of the viral PR and virion-packaged RNA.

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CROI 2015