Based deamination by CEM15 suppresses HIV-1 Infectivity

描述(申请人提供)：建议的研究是基于观察到的细胞蛋白CEM15抑制人类免疫缺陷病毒1型(HIV-1)的传染性，该病毒缺乏病毒粒子传染性因子Vif。CEM15与哺乳动物mRNA编辑酶APOBEC-1同源。据推测，CEM15抑制HIV-1的传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，而Vif抑制这种活性。具体目标1将使用CEM15的结构建模来预测将解决胞苷脱氨酶活性和抗病毒活性之间关系的位点特异性突变。这些研究将利用包括VIF在内的体外脱氨酶试验来完成，以评估其抑制脱氨酶活性的能力。病毒感染性将使用一种新的vif+和vif-HIV-1伪型慢病毒颗粒分析来量化，该方法符合CEM15目标的HIV-1区域的快速划分。特异性目标2将确定CEM15的病毒DNA或RNA底物(S)，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，并量化它们相对于野生型HIV-1的感染性，将确定观察到的DC对DU变化的生物学意义。重点还将放在对启动病毒反转录的细胞tRNAlys3引物的C到U修饰的潜在分析上。此外，一种细菌DNA错配检测系统，其中DNA修复给予包含CEM15修饰的克隆以阳性选择，将被用于识别病毒或细胞RNA或从感染细胞分离的DNA中的C到U修饰的位置。本研究重点研究CEM15的S脱氨酶活性和靶底物在抑制HIV感染性中的作用。此外，CEM15的结构建模将有助于确定潜在的Vif结合位点，并将抗病毒活性定位于CEM15的特定片段，这将有助于开发新的抗HIV-1疗法。

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.