Based deamination by CEM15 suppresses HIV-1 Infectivity

CEM15 的脱氨作用可抑制 HIV-1 感染

• 批准号: 6952761
• 负责人: Harold C Smith
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6952761
• 关键词: DNA repair; HIV infections; amination; aminohydrolases; antiAIDS agent; cell line; chemical models; cytidine; enzyme activity; microspectrophotometry; nucleic acid structure; point mutation; protein folding; protein structure function; recombinant virus; transfection; virulence; virus infection mechanism

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.

描述（由申请人提供）：拟议的研究是基于对细胞蛋白CEM15抑制缺乏病毒粒子感染因子Vif的人类免疫缺陷病毒1型（HIV-1）的传染性的观察。CEM15与哺乳动物mRNA编辑酶apobec1同源。假设CEM15抑制HIV-1感染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。Specific Aim 1将使用CEM15的结构建模来预测位点特异性突变，这些突变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测定来完成，其中包括Vif，以评估其抑制脱氨酶活性的能力。病毒传染性将使用一种新的vif +和vif- HIV-1伪型慢病毒颗粒测定法进行量化，该方法适用于CEM15靶向HIV-1区域的快速划分。特异性Aim 2将确定CEM15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突变体并量化其相对于野生型HIV-1的感染性，将确定所观察到的dC - dU变化的生物学意义。重点还将放在对启动病毒逆转录的细胞tRNAlys3引物进行C到U修饰的潜力分析上。此外，细菌DNA错配检测系统，其中DNA修复赋予含有CEM15修饰的克隆阳性选择，将用于鉴定从感染细胞分离的病毒或细胞RNA或DNA中的C到U修饰位点。本研究的重点是CEM15脱氨酶活性和靶底物在抑制HIV感染中的作用。此外，CEM15的结构建模将有助于识别潜在的Vif结合位点，并将抗病毒活性定位到CEM15的特定片段，这将有助于开发一种新的抗hiv -1治疗药物。