The proposed research is based on the observation that the cellular protein CEM15 suppresses infectivity of human immunodeficiency virus type-1 (HIV-1) that lacks the virion infectivity factor, Vif. CEM15 is homologous to the mammalian mRNA editing enzyme APOBEC-1. It is hypothesized that CEM15 suppresses HIV-1 infectivity as a direct consequence of its deaminase activity on viral or host cell nucleic acids and that Vif inhibits this activity.
Specific Aim 1 will use structural modeling of CEM15 to predict sitespecific mutants that will address the relationship between cytidine deaminase activity and anti-viral activity. These studies will be accomplished utilizing in vitro deaminase assays that will include Vif in order to assess its ability to inhibit deaminase activity. Viral infectivity will be quantified using a novel vif + and vif- HIV-1 pseudotyped lentiviral particle assay that is amenable to the rapid demarcation of regions of HIV-1 targeted by CEM15.
Specific Aim 2 will determine the viral DNA or RNA substrate(s) of CEM15 and determine the effect of Vif expression on substrate utilization. Construction of the corresponding site-specific, HIV-1 mutants and quantifying their infectivity relative to wild type HIV-1 will determine the biological significance of the observed dC to dU changes. Emphasis will also be placed on the analysis of potential of C to U modification of the cellular tRNAlys3 primer that initiates viral reverse transcription. Additionally, a bacterial DNA mismatch detection system wherein DNA repair confers a positive selection for clones containing CEM15 modifications will be employed to identify sites of C to U modification in viral or cellular RNA or DNA isolated from infected cells. This research focuses on the role of CEM15's deaminase activity and target substrate in the suppression of HIV infectivity. Moreover, the structural modeling of CEM15 will aid in the identification of potential Vif binding sites and localization of anti-viral activity to specific segments of CEM15, which will be instrumental in development of a new category of anti-HIV-1 therapeutics.
| Polevoda, Bogdan; McDougall, William M; Bennett, Ryan P et al. (2016) Structural and functional assessment of APOBEC3G macromolecular complexes. Methods 107:10-22 |
| Prohaska, Kimberly M; Bennett, Ryan P; Salter, Jason D et al. (2014) The multifaceted roles of RNA binding in APOBEC cytidine deaminase functions. Wiley Interdiscip Rev RNA 5:493-508 |
| Smith, Harold C; Bennett, Ryan P; Kizilyer, Ayse et al. (2012) Functions and regulation of the APOBEC family of proteins. Semin Cell Dev Biol 23:258-68 |
| McDougall, William M; Smith, Harold C (2011) Direct evidence that RNA inhibits APOBEC3G ssDNA cytidine deaminase activity. Biochem Biophys Res Commun 412:612-7 |
| Smith, Harold C (2011) APOBEC3G: a double agent in defense. Trends Biochem Sci 36:239-44 |
| Galloway, C A; Kumar, A; Krucinska, J et al. (2010) APOBEC-1 complementation factor (ACF) forms RNA-dependent multimers. Biochem Biophys Res Commun 398:38-43 |
| Bennett, Ryan P; Presnyak, Vladimir; Wedekind, Joseph E et al. (2008) Nuclear Exclusion of the HIV-1 host defense factor APOBEC3G requires a novel cytoplasmic retention signal and is not dependent on RNA binding. J Biol Chem 283:7320-7 |
| Bennett, Ryan P; Salter, Jason D; Liu, Xiang et al. (2008) APOBEC3G subunits self-associate via the C-terminal deaminase domain. J Biol Chem 283:33329-36 |
| Smith, Harold C (2007) Measuring editing activity and identifying cytidine-to-uridine mRNA editing factors in cells and biochemical isolates. Methods Enzymol 424:389-416 |
| Bennett, Ryan P; Diner, Elie; Sowden, Mark P et al. (2006) APOBEC-1 and AID are nucleo-cytoplasmic trafficking proteins but APOBEC3G cannot traffic. Biochem Biophys Res Commun 350:214-9 |
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