CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

191 HIV-1 Nef Dimerization Is Important for AP2 Recruitment and CD4 Downregulation Sherry Shu ; Lori A. Emert-Sedlak;Thomas E. Smithgall Univ of Pittsburgh, Pittsburgh, PA, USA

Background: The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef hijacks host cell trafficking pathways to downregulate membrane-bound receptors, including CD4 via the adaptor protein 2 (AP2) complex. CD4 downregulation is linked to enhanced viral infectivity and immune escape, identifying this important Nef function as a rational target for new antiretroviral drug discovery. Methods: We developed a cell-based bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of Nef with the AP2 complex and CD4. Interacting protein pairs were fused to complementary, non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with CD4 or AP2 result in complementation of YFP and a bright fluorescent signal by confocal microcopy. Results: Nef interaction with both CD4 and the AP2 α and σ2 subunits was readily visualized in live cells by BiFC assay. Co-expression of the AP2 α subunit enhanced the Nef:AP2 σ2 BiFC signal and vice versa, suggesting that the AP2 α/σ2 hemi-complex interacts cooperatively with Nef. Mutagenesis of Nef residues R134, E174, and D175, which stabilize the Nef docking surface for AP2 in a recent co-crystal structure (PDB: 4NEE), substantially reduced AP2 interaction without affecting CD4 binding. Remarkably, a dimerization-defective mutant of Nef failed to interact with either CD4 or AP2 in the BiFC assay, indicating that the quaternary structure of Nef is required for CD4 and AP2 recruitment as well as CD4 downregulation. Small molecule inhibitors of Nef dimerization also reduced Nef interactions with AP2 and CD4 by BiFC, providing further support for this idea and identifying the Nef dimer interface as a target for inhibitor action. Conclusions: Here we describe a fluorescence complementation assay to visualize interactions between Nef, CD4 and the AP2 complex in live cells. Using this assay, we observed that the AP2 α and σ2 subunits interact cooperatively with Nef, and that the interaction is dependent upon an internal Nef salt bridge present in the recent crystal structure of the Nef:AP2 complex. Complex assembly also requires an intact Nef dimerization interface, consistent with previous observations that dimerization-defective Nef mutants fail to downregulate CD4. Small molecules that inhibit Nef dimerization also suppress AP2 and CD4 recruitment, which may provide a mechanistic explanation for their effects on HIV infectivity. 192 Quantification of HIV-1 Splicing Using a PrimerID-Tagged Deep Sequencing Assay Ann Emery ; Ronald I. Swanstrom Univ of North Carolina at Chapel Hill, Chapel Hill, NC, USA Background: HIV-1 RNA is spliced to produce more than 40 different transcripts. Most previous research on HIV-1 splicing used model single exon systems and PCR gel quantification. Our straightforward assay quantifies HIV-1 RNA splicing patterns in the context of viral infection using Illumina paired-end deep sequencing and PrimerID. This system can be used to assess changes in splicing profiles by mutations that affect splice regulatory elements and to assess the differential effect of APOBEC3G-mediated mutagenesis on the 4 kb partially spliced mRNA population compared to the 1.8 kb mRNA population. Methods: PrimerID is a random sequence tag in the cDNA primer, which is incorporated into a cDNA and all PCR products subsequently made from that tagged cDNA. Each cDNA gets a unique PrimerID and after sequencing, the PCR products can be sorted by their PrimerIDs. Each occurrence of a PrimerID is counted exactly once, and skewing in the PCR steps is filtered out. Additionally, a consensus sequence can be made frommultiple reads with the same PrimerID, making it possible to filter out PCR and sequencing errors. PrimerID-tagged reverse primers specific for either the 1.8kb or 4kb splice classes and a common forward primer were used to tag the 40+ HIV-1 spliced mRNAs. Following amplification and Illumina paired-end deep sequencing, mRNAs were quantified according to splice type and collapsed into consensus sequences. Mutations in a stem near A1 and in the ESE(GAR) sequence located between A5 and D4 were correlated with splicing outcomes. Results: Mutations to a highly conserved stem-loop containing splice acceptor A1 caused a 10-fold reduction in A1 usage, resulting in reduced vif transcript levels. The ESE(GAR) splice control element between A5 and D4 was analyzed for APOBEC3G mutations. Reduced vif transcripts led to increased C=>T mutations, which were unevenly skewed towards the 1.8kb splice class. T=>C mutations, though less frequent, skewed towards the 4kb splice class. As this panel of mutations in the ESE(GAR) region illustrates, C=>T and T=>C transitions differentially affect upstream (A5) splicing and/or downstream (D4-A7) splicing. Conclusions: Combining PrimerID with paired-end deep sequencing allows mutations in a splice control element to be screened and correlated to real-time changes in HIV-1 splicing. We identified mutations near A1 and between A5 and D4 that affect utilization of the proximal splice sites.

Poster Abstracts

193

Effects of Differential Cell Signaling in HIV Transcription Matthew J. Gagne 1 ;WilsonW.Wong 2 ; Gillian M. Schiralli Lester 3 ; Andrew Henderson 1 1 Boston Univ Sch of Med, Boston, MA, USA; 2 Boston Univ, Boston, MA, USA; 3 Univ of Rochester Med Cntr, Rochester, NY, USA

Background: After cessation of antiviral treatments, HIV viral levels rebound due to the existence of latent reservoirs. The elucidation of signals that bias a provirus towards a latent or robust infection is necessary for more thorough treatment of HIV/AIDS patients. We propose a model system by which signals from CD3 and CD28, receptors necessary for T cell activation, can be manipulated at the time of infection of CD4+ T cells. This allows us to alter the balance between productive infection and the establishment of latent reservoirs.

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CROI 2016