CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

172 CHARACTERIZING APOBEC3H FUNCTION AND EVOLUTION IN AFRICAN GREEN MONKEYS Erin I. Garcia 1 , Michael Emerman 2 1 Univ of Washington, Seattle, WA, USA, 2 Fred Hutchinson Cancer Rsr Cntr, Seattle, WA, USA

Background: Members of the APOBEC3 family of genes are cytidine deaminases involved in innate immune defense against retroviruses and retroelements. One member of the family, APOBEC3H (A3H), is polymorphic in humans. These polymorphisms have been demonstrated to impact not only the stability of the protein, but also affects interactions with its viral antagonist, HIV-1 Vif. We hypothesized that this mechanism of polymorphismmay be evolutionarily conserved. Therefore, we characterized A3H variation within African Green Monkeys (AGM) in order to further elucidate the dynamics of the A3H-Vif interaction in this primate model. Methods: A3H was amplified and sequenced from a panel of AGMs across all four subspecies: vervet, tantalus, sabaeus, and grivet. Phylogenetic analyses were conducted on nucleotide and protein sequences. The ability of haplotypes to restrict viral infection by SIVagmwas tested in single cycle infections. Results: We found that A3H is extraordinarily polymorphic in African Green Monkeys (AGM), which provides a unique opportunity to study the interaction of A3H haplotypes with four species specific lentiviruses. We identified a total of 35 polymorphisms in A3H of AGMs. 25 of these polymorphisms are nonsynonymous and occur across all four subspecies of AGM. Many polymorphisms are tightly linked in one region of the gene, which results in a bifurcation of the phylogenetic tree. Haplotypes that encode either a CGRELP or SRQKRQ motif within this region group together within a possible Vif-binding interface. Additionally, two species specific amino acid changes have occurred in this same region in vervet monkeys. Furthermore, a subset of haplotypes have been tested for antiviral activity in single-round infectivity assays and also demonstrate antiviral activity against SIVagm from other subspecies. The affects of this polymorphism on A3H-Vif binding interactions and virus replication and evolution are being studied. Conclusion: Polymorphism in A3H has impacted HIV dynamics in humans, but the interaction between A3H and HIV-1 Vif is not yet fully understood. We have shown that A3H is also polymorphic in another primate model. These polymorphisms may impact A3H-Vif interactions in AGMs and would provide another forum to identify factors that are important in such interactions. 173 MAPPING THE HIV-1 VIF-CBFβ INTERACTION INTERFACE AND IDENTIFICATION OF DETERMINANTS Belete A. Desimmie 1 , Jessica Smith 1 , Hiroshi Matsuo 2 , Wei-Shau Hu 1 , Vinay K. Pathak 1 1 NCI, Frederick, MD, USA, 2 Univ of Minnesota, Minneapolis, MN, USA Background: Human APOBEC3 (A3) proteins such as A3G are host restriction factors that deaminate cytidines in DNA and potently inhibit HIV-1 replication by inducing lethal G-to-A hypermutation. However, HIV-1 encodes the accessory protein viral infectivity factor (Vif), which counteracts the antiviral activities of A3 proteins. Vif targets the A3 proteins for proteasomal degradation by recruiting components of an E3-ubiquitin ligase complex. Previous studies have demonstrated that Vif hijacks the cellular transcription co-activator core-binding subunit beta (CBFβ) to mediate A3G degradation and have identified different residues of Vif involved in Vif-CBFβ interaction. Recently a co-crystal structure of HIV-1 Vif, CBFβ, Cul5, EloB, and EloC pentameric complex was solved. However, a systematic analysis of the functional importance of the Vif-CBFβ interaction interface in cells has not been determined, and the amino acids involved in the interaction in cells have not been fully characterized. Methods: To identify the critical Vif-CBFβ interaction determinants, we performed double-alanine scanning mutagenesis of the first 60 amino acids of Vif. We then determined the interaction efficiencies of the different Vif mutants with CBFβ by immunoprecipitation as well as the biological significance of these interactions by examining A3G degradation and viral infectivity. Results: We found that multiple Vif residues are involved in the extensive N-terminal Vif-CBFβ interaction; particularly, 5 WQVMIVW 11 region of Vif was found to be the major determinant. A minimum of three alanine substitutions were required to completely abrogate the Vif-CBFβ interaction. Furthermore, these mutants were unable to rescue HIV-1 infectivity in the presence of A3G. A reciprocal mutational analysis targeting CBFβ revealed that F68 and I55 residues are important and participate in a tripartite hydrophobic interaction to maintain a stable and functional Vif-CBFβ complex. Conclusion: Our results provide detailed insight into the major determinants of interaction between Vif and CBFβ in cells. Together with the available structural data of the Vif-CBFβ -E3 ubiquitin ligase pentameric complex, our data provide valuable information for future structure-based rational design of a novel class of HIV-1 inhibitors targeting Vif-mediated degradation of A3 proteins. 174 THE ABILITY OF HIV-1 TO EVADE SAMHD1-MEDIATED RESTRICTION IN MACROPHAGE Timothy Plitnik , Mark Sharkey, Mario Stevenson Univ of Miami, Miami, FL, USA Background: SAMHD1 has been described as an antiviral restriction factor that functions by reducing intracellular dNTP pools, thus creating hostile conditions for reverse transcription. SIV and HIV-2 encode Vpx, which promotes the degradation of SAMHD1 thereby increasing the cellular dNTP pool. Although HIV-1 lacks Vpx, it readily infects cells with SAMHD1 activity and low dNTP levels such as macrophage. Therefore, it is unclear how HIV-1 avoids antagonism by SAMHD1. We hypothesize that reverse transcriptase of HIV-1 has a lower Km for dNTPs, which allows it to overcome SAMHD1 restriction without Vpx. Methods: Elutriated human monocytes were differentiated into macrophage with MCSF for 7 days. Cells were then infected with HIV-1 or SIV (+/- Vpx) normalized by Gag ELISA. SAMHD1 levels post-infection were quantified by FACS analysis and western blots. RNAi was used to knock-down SAMHD1 in macrophage, which were subsequently used for infection. GFP-expressing HIV-1 or SIV was used in pre-infection experiments, which were also quantified by FACS analysis. Chimeric viruses were generated using Exponential- Megapriming-PCR. Reverse transcription was quantified using qPCR for 2LTR circles normalized to genomic CCR5 copy number. Results: We first demonstrate that HIV-1 can infect macrophage without affecting SAMHD1 levels. However, SIV reverse transcription is dependent on SAMHD1 degradation. RNAi knock-down of SAMHD1 enables SIVΔVpx infection in macrophage. Pre-infection of macrophage by HIV-1 was not sufficient to rescue a SIVΔVpx, revealing that HIV-1 is not altering the intracellular environment, and its ability to overcome SAMHD1 activity is intrinsic to the virus itself. Chimeric viruses with exchanged RTs were created and used to test the hypothesis. We found that the SIVΔVpx was not able to overcome restriction with an HIV-1 RT and that an SIV RT did not render HIV-1 susceptible to restriction. Conclusion: HIV-1 has evolved to infect myeloid cells without Vpx, whereas SIV is highly dependent upon Vpx-mediated SAMHD1 degradation. Exchange of the RT’s demonstrated that HIV-1 RT was not able to overcome SAMHD1 restriction in SIVΔVpx, nor did the SIV RT render HIV-1 susceptible to restriction. These findings indicate that kinetic differences in the RT’s are not sufficient to confer the ability of HIV-1 to infect macrophage without Vpx. HIV-1 escape from SAMHD1 restriction is dependent on determinants distinct from RT. 175 MHC DOWN-MODULATION IN HIV-INFECTED CELLS OCCURS LATE IN THE VIRUS REPLICATIVE CYCLE Rodrigo Matus Nicodemos , Daniel Douek, Richard A. Koup Vaccine Rsr Cntr, NIAID, Bethesda, MD, USA Background: In vitro studies suggest that HIV-1 Nef selectively down-modulates HLA-A and HLA-B molecules to protect the infected cells from CD8 T cell-mediated recognition and killing. However, the emergence of escape mutations in vivo clearly shows that CD8 T cells must exert selective pressure though recognition of virus epitopes presented by the MHC class I molecules. We hypothesized that virus peptide-MHCs are presented during the virus replicative cycle before Nef completely down-modulates MHC molecules. Therefore, HIV-infected cells can be recognized and eliminated by HIV-specific CD8 T cells. Methods: To test this hypothesis we developed a FACS-based virus replicative cycle reporter system to measure the timing of MHC down-modulation. A GFP IRES Nef construct was inserted into the Nef ORF of the NL4-3 replication competent molecular clone. PHA/anti-CD28-activated primary CD4 T cells were infected for 6 hours, washed, and further incubated for 24 or 48 hours. The early stage of the virus replicative cycle was detected by the expression of GFP while the late stage was detected by intracellular staining with an anti-HIV Gag p24 antibody. We used antibodies specific to HLA-A02, A03, B07, B08, and B27 to determine the timing of the down-modulation for these molecules. As controls we

Poster and Themed Discussion Abstracts

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CROI 2017