CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

are unknown. The structure of SIVmac Vpx bound to DCAF1 and the C-terminus of SAMHD1 has been solved, but there is extreme sequence diversity in vpr and vpx from divergent viruses. Methods: We used an evolutionary and structural approach to find appropriate and robust breakpoints in Vpx and Vpr in order to create functional, chimeric viral proteins. By assaying for the gain of ability to degrade resistant SAMHD1, these chimeric proteins assisted in mapping of determinants of specificity in Vpx and Vpr from several lentiviral lineages. Results: We found that the majority of residues involved in binding DCAF1 were conserved in essentially all SAMHD1-degrading Vpx and Vpr. We identified highly conserved amino acids flanking the regions of SIVmac Vpx involved in binding SAMHD1. These conserved motifs served as breakpoints to create chimeric proteins between Vpr and Vpx from SIVs infecting macaque, red-capped mangabey, and African green monkeys. We were then able to retarget Vpx and Vpr from different lentiviruses to degrade heterologous SAMHD1. Depending on the lineage, the specificity of Vpx/r in degrading SAMHD1 maps to one or two regions in the viral protein. Conclusions: The structure of Vpx and Vpr proteins and their interaction with the host ubiquitin ligase is widely conserved, despite high levels of sequence variation across lineages. Given the evolutionary constriction in maintaining this ubiquitin ligase binding, similar regions of Vpr/x are used to target SAMHD1, allowing the mapping of sites that govern SAMHD1 antagonism in species-specific interactions. 202 A Surprising New Function of SAMHD1 as a Pro-Pathogenic Factor in HIV Infection Background: Depletion of CD4 T cells and development of chronic inflammation are signature processes in HIV pathogenesis that propel disease progression. Due to endogenous SAMHD1 restriction activity in quiescent lymphoid CD4 T cells, the viral chain elongation phase of reverse transcription is attenuated, giving rise to incomplete cytosolic DNA transcripts. CD4 T-cell death is triggered after sensing of these cytosolic DNA intermediates by interferon gamma Inducible protein 16 ( IFI16 ). Death occurs following caspase-1 activation in inflammasomes and the induction of pyroptosis , a highly inflammatory form of programmed cell death. These findings mechanistically connect CD4 T-cell death and chronic inflammation––the two signature pathogenic processes of active HIV infection. Methods: Human lymphoid aggregated cultures (HLACs) prepared using tonsil and spleen, and lymph node biopsies from consenting HIV-infected volunteers were used. Results: We now show that SAMHD1 restriction activity influences how CD4 T cells die. Degradation of SAMHD1 by Vpx encoded by HIV-2 thwarts abortive infection in resting, non-permissive lymphoid CD4 T cells redirecting the cell death pathway away from caspase-1-mediated pyroptotic pathway (inflammatory) toward caspase-3-mediated apoptotic pathway (noninflammatory). SAMHD1 effectively suppresses caspase-1 activation and pyroptosis when infection occurs with cell-free virions. However, in the context of cell-to- cell transmission, which is 100-1,000-fold more efficient, SAMHD1 restriction is only partially effective resulting in the accumulation cytoplasmic viral DNA. This DNA is sensed by IFI16 resulting in caspase-1 activation and triggering of the pyroptotic death pathway. The action of other cellular factors like TREX1 and SLX4 (single and double strand DNA nucleases) may require further increase in the levels the cytosolic DNA needed to trigger pyroptosis. Conclusions: 1. The Vpx protein thwarts inflammatory pyroptosis following HIV-2 infection by degrading SAMHD1 thereby avoiding abortive infection and sensing of cytosolic viral DNA. 2. SAMHD1 is a bifunctional host factor capably restricting infection of resting CD4 T cells by cell-free HIV virions but functioning as a pro-pathogenic factor when resting CD4 T cells are infected by HIV-1 by the cell-to-cell route. 191 Characterization of the Activity of an Innate Immunity Protein, the Apolipoprotein L6 Nitisha Pyndiah 1 ; Angela Ciuffi 1 ; AmalioTelenti 2 1 Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 2 J Craig Venter Institute, San Diego, CA, US Background: Host restriction factors are proteins hampering virus replication, and share common features including positive selection, viral counteraction, interferon-inducible expression and differential expression among HIV+ patients. APOL6 was identified in a screen aiming at identifying novel HIV-1 candidate host restriction factors. Another member of the APOL family, APOL1, has been previously described for its protective role against the parasite Trypanosoma brucei and more recently as a new HIV restriction factor (Taylor et al., 2013). The aim of the study was to characterize the activity of APOL6. Methods: We evaluated the ability of APOL6 primate orthologues, chimeras and mutants to inhibit the GFP expression from an HIV-1 based genomic vector in co-transfection experiments. We also assessed the impact of APOL6 on transduction of reporter viruses. Analysis was carried out using flow cytometry Results: APOL6-mediated restriction was validated in a co-transfection assay with an HIV-1 LTR-EF1-GFP, showing up to 10-fold reduced GFP expression in APOL6-expressing cells compared to control cells. Species-specific restriction of APOL6 primate orthologs co-transfected with HIV-1 LTR-EF1-GFP revealed a higher GFP inhibition mediated by hominids and new world monkey APOL6 compared to old world monkeys APOL6. A similar APOL6-mediated inhibition was observed when APOL6 was co-transfected with alternate GFP expression vectors. In contrast, APOL6 was not able to restrict HIV-based vector transduction nor adenovirus or LCMV. Furthermore, APOL1 and APOL3 constructs were also tested in co-transfection and transduction experiments and followed the same tendency as APOL6. Through co-transfection analyses using human and rhesus APOL6, respectively displaying high and low inhibition ability, we identified a specific APOL6 domain and residue mediating APOL6 effects. Conclusions: All together, these data suggest that APOL6-mediated activity is not virus-specific, but rather displayed a broad action against various promoter constructs. This points out to a specific APOL6 mechanism, potentially acting at the level of nucleic acid (DNA or RNA) sensing and/or degradation. APOL6 domain and residue responsible for the activity was identified. The mechanism used by APOL6 is very likely to be shared by other members of the family. We are currently identifying APOL6 cellular interactants by Mass spectrometry to elucidate the mechanism of APOL6-mediated restriction. 531 Pharmacokinetic Interactions Between Antidiabetics and Efavirenz Using PBPK Modeling Catia Marzolini 1 ; Rajith Rajoli 2 ; Luigia Elzi 1 ; Manuel Battegay 1 ; David Back 2 ; Marco Siccardi 2 1 University Hospital Basel, Basel, Switzerland; 2 Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom Background: Diabetes has emerged as an important co-morbidity in the aging HIV population. The management of diabetes is complicated by the issue of drug-drug interactions (DDI) between antidiabetics and antiretroviral drugs and the lack of clinical data on how to manage these DDI. The antidiabetics pioglitazone (PIO) and repaglinide (REP) are metabolized by CYP2C8 and CYP3A4 and therefore are subject to DDI with efavirenz (EFV), an inducer of CYP3A4 and inhibitor of CYP2C8. The objective of this study was to simulate the pharmacokinetic (PK) interaction between PIO or REP and EFV using physiologically based pharmacokinetic (PBPK) modeling. Methods: In vitro data describing the physicochemical properties, absorption, distribution, metabolism and elimination of PIO, REP and EFV, as well as the CYP induction and inhibition potential of EFV were obtained from published literature. The experimental data were integrated in a PBPK model developed using Simbiology (Matlab, R2013b), Gilad Doitsh ; Nicole Galloway; Xin Geng; Isa Monus Arias; ZhiyuanYang;Warner C. Greene The J. David Gladstone Institute, University of California San Francisco, San Francisco, CA, US

Oral Abstracts

142

CROI 2015