CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

199 Transcriptional Regulation of the APOBEC3 Repertoire Through the CBFß/RUNX Axis Brett D. Anderson 1 ; Reuben S. Harris 2 1 Univ of Minnesota, Minneapolis, MN, USA; 2 Howard Hughes Med Inst, Minneapolis, MN, USA

Background: Several members of the APOBEC3 family of DNA cytosine deaminases have the capacity to potently restrict HIV-1 replication by inducing extensive G-to-A mutations in the viral cDNA during reverse transcription. HIV-1 overcomes this antiviral defense by encoding the small protein Vif, which recruits a cellular ubiquitin ligase to target the APOBEC3 proteins for degradation through a polyubiquitination mechanism. Recently, we identified the cellular transcription factor CBFβ as an obligate Vif binding partner and essential component of this ubiquitin ligase. CBFβ normally forms a heterodimer with members of the RUNX family of DNA binding transcription factors to regulate expression of numerous genes involved in hematopoietic development and immune function. A recent structural study on the Vif-CBFβ complex revealed that Vif engages a surface on CBFβ that overlaps significantly with the known RUNX binding surface. Therefore, we hypothesized that the viral hijacking of CBFβ to promote APOBEC3 degradation may concurrently promote viral replication by directly impeding the assembly of functional CBFβ/RUNX heterodimers, ultimately resulting in altered expression of cellular genes. Methods: A combination of shRNA knockdown and CRIPSR/Cas9 gene disruption approaches were employed to deplete CD4+ T cells of endogenous CBFβ. CBFβ-dependent changes in cellular gene expression were measured by immunoblotting and RT-qPCR, and HIV-1 replication kinetics in CBFβ-depleted cell lines were assayed in single-cycle and spreading infection experiments. Results: Surprisingly, we found that CBFβ functions in complex with the RUNX proteins as positive regulators of the APOBEC3 genes themselves in CD4+ T cells, and that genetic knockdown and knockout of CBFβ is sufficient to render T cells permissive to Vif - deficient HIV-1 replication due to a loss of antiviral APOBEC3 gene expression. Conclusions: Based on these results, we propose a two-pronged model whereby HIV-1 Vif counteracts the APOBEC3 antiviral defense by directly promoting APOBEC3 polyubiquitination while simultaneously suppressing APOBEC3 gene transcription by sequestering CBFβ from RUNX-associated transcription complexes. 200 Is Endogenous APOBEC3H an HIV-1 Transmission Barrier? Jiayi Wang 1 ; Allison M. Land 1 ; Brian J. Hoium 1 ; EricW. Refsland 1 ; Elizabeth M. Luengas 1 ; Romel D. Mackelprang 2 ;William L. Brown 1 ; Michael Emerman 3 ; Jairam R. Lingappa 2 ; Reuben S. Harris 4 1 Univ of Minnesota, Minneapolis, MN, USA; 2 Univ of Washington, Seattle, WA, USA; 3 Fred Hutchinson Cancer Rsr Cntr, Seattle, WA, USA; 4 Howard Hughes Med Inst, Minneapolis, MN, USA Background: Several members of the APOBEC3 family of DNA cytosine deaminases can potently inhibit HIV-1 replication by catalyzing extensive cytosine deamination in viral cDNA during reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which adapts the APOBEC3 proteins to a cellular ubiquitin ligase to target them for proteasomal degradation. In humans, A3H is the most polymorphic member of the family and includes seven haplotypes with three encoding for stable proteins and the rest unstable. Stable A3H proteins contribute to HIV-1 restriction and can only be counteracted by hyper-functional but not hypo-functional Vif variants (dictated by amino acids at key positions). We hypothesize stable A3H enzymes provide a natural barrier to HIV-1 acquisition from patients with unstable A3H. Methods: A3H haplotypes of 597 serodiscordant couples with both transmission and non-transmission events were determined from blood spot genomic DNA using novel quantitative PCR assays. The vif genotype for each patient was determined by sequencing RT-PCR products from patient serum samples. Consensus vif open reading frames were cloned into expression vectors and assayed against stable A3H in single-cycle infectivity experiments. Results: HIV-1 restriction by stable A3H can only be counteracted effectively by hyper-functional Vif with F39, H48, and EKGE60-63. In the serodiscordant couples cohort, the transmission rate from index patients with unstable A3H to partners with stable A3H is 34%, which is the same as the overall transmission rate of all analyzed couples. Vif genotyping of the index patients within the unstable-to-stable subgroup show that the majority (65%) of the Vif variants are predicted to be hyper-functional and resistant to stable A3H. Conclusions: Endogenous stable A3H proteins restrict infection by HIV-1 with hypo-functional Vif variants in primary T lymphocytes. However, on population level, stable A3H is not a protective factor in HIV-1 acquisition from individuals with unstable A3H. Nonetheless, natural resistance to infection is likely to be due to many distinct mechanisms, stable A3H enzymes may provide a transmission barrier against HIV-1 isolates harboring hypo-functional Vif alleles. 201 Characterization of Novel Human APOBEC3A Variants Matthew Hernandez ; Lara Manganaro; Marcel Ooms; Lubbertus C. Mulder;Veronica Iannucci; Marsha Dillon-White; Sandra N.Terry; Ekta Sharma;Viviana A. Simon Icahn Sch of Med at Mount Sinai, New York, NY, USA Background: The APOBEC3 cytidine deaminases (A3A to A3H) are part of the host’s intrinsic defense against retroviruses. The human APOBEC3 locus has rapidly expanded through multiple gene duplication events: mice only encode a single APOBEC3 gene while primates have seven different APOBEC3 proteins. Such genomic regions are often hot spots for structural genomic variation. A common somatic structural variant deletes the 3’UTR of A3A and the coding region of A3B. The impact of this copy number variation on A3A transcripts is currently not well understood. We speculate that the loss of the A3B coding region results in novel A3A splice variants and/or A3A-A3B chimeras. Because A3A has been implicated in the restriction of HIV as well as HTLV-1, we hypothesize that novel A3A variants could have a significant impact on viral replication and pathogenicity Methods: A3B copy number variation was determined by real-time qPCR from peripheral blood lymphocytes of 156 HIV negative blood donors. A3A and A3A-A3B chimera transcripts were amplified, cloned and sequenced of a subset of donors lacking A3B (N=3) or carrying one copy (N=5), two copies (N=2), or three A3B copies (N=1). Wild-type A3A and novel A3A variants were cloned into a mammalian expression vector. The catalytic activity of A3A was assessed by co-transfecting each A3A variant with a plasmid expressing a blue fluorescent protein (BFP) followed by flow cytometry analyses. The cellular localization of the different splice variants was determined by confocal microscopy. Results: We cloned and sequenced 112 A3A transcripts from 11 donors that differ in A3B copy number. We identified novel A3A splice variants and detected A3A-A3B chimeric transcripts from cells lacking one or both A3B copies. These A3A variants resulted in longer and shorter variants and one A3A variant acquired 50 unique residues compared to the 199 amino acid long A3A reference. The catalytic deaminase activity of the A3A variants was significantly affected by certain A3A variants when compared to the reference A3A. Confocal microscopy revealed that the reference A3A and most A3A variants have a pan-cellular localization, whereas a shorter A3A variant exclusively shows a perinuclear localization. Conclusions: We found that the A3B deletion polymorphism in the APOBEC3 locus results in novel A3A and A3A-A3B chimeric transcript variants. These A3A variants have profound effects on catalytic activity and cellular localization and we are currently characterizing their antiviral effects. 202 CBFß Protects HIV-1 Vif FromMDM2-Mediated Degradation Yusuke Matsui ; Keisuke Shindo; Kayoko Nagata; NoriyoshiYoshinaga; Kotaro Shirakawa; Masayuki Kobayashi; AkifumiTakaori-Kondo Kyoto Univ, Kyoto, Japan Background: HIV-1 overcomes the host restrictive APOBEC3 (A3) proteins by organizing an E3 ubiquitin ligase complex together with Vif and a host transcription factor CBFβ. CBFβ is essential for Vif function by increasing steady-state level of Vif protein and enabling the ubiquitin ligase recruitment, however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Since we have previously reported that murine double minute 2 homolog (MDM2) is an E3 ligase for Vif protein, we hypothesized that CBFβ might protect MDM2-mediated degradation of Vif. Methods: To test whether CBFβ interferes with the interaction between Vif and MDM2, co-immunoprecipitation of MDM2 with Vif derivatives that do not bind CBFβ was performed. To ask whether MDM2 is relevant to Vif metabolism, MDM2 -null cells were used for knockdown by siRNA against CBFβ and for cycloheximide chase experiments of one of the Vif derivatives. To identify Vif residue that interacts with MDM2, Vif-degradation assays, co-immunoprecipitation and single-cycle infection of HIV were performed with Vif amino acid substitution derivatives.

Poster Abstracts

78

CROI 2016