Based deamination by CEM15 suppresses HIV-1 Infectivity

描述（由申请方提供）：拟定研究基于细胞蛋白CEM 15抑制缺乏病毒体感染因子Vif的人类免疫缺陷病毒1型（HIV-1）感染性的观察结果。CEM 15与哺乳动物mRNA编辑酶APOBEC-1同源。假设CEM 15抑制HIV-1感染性是其对病毒或宿主细胞核酸的脱氨酶活性的直接结果，并且Vif抑制该活性。Specific Aim 1将使用CEM 15的结构建模来预测位点特异性突变体，这些突变体将解决胞苷脱氨酶活性和抗病毒活性之间的关系。这些研究将利用体外脱氨酶试验完成，该试验将包括Vif，以评估其抑制脱氨酶活性的能力。 将使用新型vif +和vif-HIV-1假型慢病毒颗粒测定法定量病毒感染性，该测定法适用于快速划分CEM 15靶向的HIV-1区域。特定目标2将确定CEM 15的病毒DNA或RNA底物，并确定Vif表达对底物利用的影响。构建相应的位点特异性HIV-1突变体并定量其相对于野生型HIV-1的感染性将确定所观察到的dC至dU变化的生物学意义。重点还将放在分析启动病毒逆转录的细胞tRNAlys 3引物的C至U修饰的潜力上。此外，将使用细菌DNA错配检测系统来鉴定病毒或细胞RNA或从感染细胞分离的DNA中的C至U修饰位点，其中DNA修复赋予对含有CEM 15修饰的克隆的阳性选择。本研究的重点是CEM 15的脱氨酶活性和靶底物在抑制HIV感染性中的作用。此外，CEM 15的结构建模将有助于鉴定潜在的Vif结合位点和将抗病毒活性定位于CEM 15的特定片段，这将有助于开发新类别的抗HIV-1治疗剂。

DESCRIPTION (provided by applicant): 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.