RNA Aptamers Targeted to HIV-1 PR, Vif and Nef Proteins

针对 HIV-1 PR、Vif 和 Nef 蛋白的 RNA 适体

• 批准号: 7495629
• 负责人: Sonald Duclair
• 金额: $ 5.33万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-16 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7495629
• 关键词: Affinity; Binding; Biochemical; Cell Line; Cells; Cis-Acting Sequence; Collaborations; Condition; Cultured Cells; Dissociation; Endopeptidases; Enzymes; Escape Mutant; Evolution; Gel; Genome; Genomics; Goals; HIV; HIV Infections; HIV-1; HIV-1 protease; Hematopoietic; In Vitro; Infection; Letters; Ligands; Matrix Metalloproteinases; Mutation; Peptide Hydrolases; Process; Property; Proteins; Publishing; RNA; RNase protection assay; Research; Retroviral Vector; Testing; Texas; Transcript; Universities; Viral; Virion; Virus; aptamer; day; mutant; nef Protein; particle; pressure; prevent; promoter; research study; resistance mechanism; virus culture

DESCRIPTION (provided by applicant): The main goal of the proposed research is to isolate efficacious RNA aptamers against one HIV-1 enzyme and two accessory proteins: PR, Vif and Nef, and to delineate their efficacy and mode of action in blocking viral replication. SELEX experiments will be performed to generate aptamers against these currently unexplored HIV targets. Aptamers will be cloned, sequenced and subjected to additional screens for optimal expression levels and Compartmentalization in cells susceptible to HIV infection. Experiments will be performed in vitro to determine their dissociation constant and ability to inhibit functions of their protein targets. Six of the tightest binding aptamers in each case will be expressed via U6+1 promoter on the retroviral vector MMP-eGFP and used to tranduce CEM x 174 cell lines. These cells will be subjected to virus challenge experiments. In addition, several target-specific experiments will be performed to delineate the mode and efficacy of inhibition employed by these aptamers. Selection experiments will also be performed to identify functional mutations in the target proteins that can escape aptamer pressure. In addition, several experiments will be conducted in an attempt to further optimize these RNA aptamers.

描述（由申请人提供）：拟议研究的主要目标是将有效的RNA适体与一种HIV-1酶和两种辅助蛋白分离出来：PR，VIF和NEF，并描述其在阻断病毒复制方面的功效和作用方式。 将执行SELEX实验，以生成针对这些目前未开发的HIV目标的适体。 适体将被克隆，测序并接受其他筛选，以使其在容易受到HIV感染的细胞中以最佳的表达水平和分隔。 实验将在体外进行，以确定其解离常数和抑制其蛋白质靶标功能的能力。 在每种情况下，最紧密的结合适体中的六个将通过逆转录病毒载体MMP-EGFP上的U6+1启动子表示，并用于将CEM CEM X 174细胞系替换。 这些细胞将受到病毒挑战实验。 此外，将进行几项针对特定目标的实验，以描述这些适体采用的抑制作用的模式和疗效。 还将进行选择实验，以鉴定靶蛋白中可以避免适体压力的功能突变。 此外，将进行一些实验，以进一步优化这些RNA适体。