Based deamination by CEM15 suppresses HIV-1 Infectivity

CEM15 的脱氨作用可抑制 HIV-1 感染

• 批准号: 6746465
• 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 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6746465
• 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）：拟议的研究基于以下观察结果：细胞蛋白CEM15抑制了缺乏病毒体感染因子VIF的人类免疫缺陷病毒类型1（HIV-1）的感染性。 CEM15与哺乳动物mRNA编辑酶APOBEC-1同源。假设CEM15抑制HIV-1感染性是其在病毒或宿主细胞核酸上的脱氨酶活性的直接结果，并且VIF抑制了这种活性。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地点突变体。这些研究将利用包括VIF在内的体外脱氨酶测定法完成，以评估其抑制脱氨酶活性的能力。 将使用新型的VIF +和VIF-HIV-1假病毒颗粒测定法对病毒感染性进行量化，该测定与CEM15靶向的HIV-1区域的快速划分。具体目标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。相应的位点特异性，HIV-1突变体的构建和相对于野生型HIV-1量化其感染力将确定观察到的直流对DU变化的生物学意义。还将重点放在C的电势分析对启动病毒逆转录的细胞TRNALYS3底漆的U型的分析上。此外，将采用一个细菌DNA不匹配检测系统，其中DNA修复将对含有CEM15修饰的克隆进行阳性选择，以识别从感染细胞分离的病毒或细胞RNA或DNA中C到U修饰的位点。这项研究的重点是CEM15的脱氨酶活性和靶标底物在抑制HIV感染性中的作用。此外，CEM15的结构建模将有助于鉴定潜在的VIF结合位点，并将抗病毒活性定位到CEM15的特定段，这将在开发新类别的抗HIV-1治疗疗法方面发挥作用。