Biochemical Mechanism of HIV DNA Integration

HIV DNA整合的生化机制

• 批准号: 6948390
• 负责人: Alan N. Engelman
• 金额: $ 42.75万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6948390
• 关键词: DNA binding protein; DNA footprinting; RNA interference; chromatin immunoprecipitation; clinical research; genetic promoter element; host organism interaction; human immunodeficiency virus 1; human tissue; integrase; laboratory mouse; polymerase chain reaction; protein purification; protein structure function; site directed mutagenesis; virus DNA; virus infection mechanism; virus integration; virus protein; western blottings

DESCRIPTION (provided by applicant): Replication of human immunodeficiency virus type 1 (HIV-1) causes AIDS in humans, and antiviral drugs developed by the pharmaceutical industry are currently the most effective way to combat HIV/AIDS. HIV-1 makes three of its own enzymes, reverse transcriptase, protease, and integrase that are essential for its growth. Drugs currently in the clinic target reverse transcriptase and protease, and therefore integrase is the only viral enzyme that is currently untargeted by clinicians. To learn more about the mechanism of integrase action, this application proposes to decipher how the enzyme functions to integrate its cDNA substrate during infection in human cells. During infection, the virus forms a functional preintegration complex comprised of integrase, cDNA, and other viral as well as human cell proteins. Numerous different human proteins have been implicated in the function of HIV-1 preintegration complexes, but it is unclear to what extent these proteins are required during infection. A variety of protein biochemistry and viral genetic experiments will be performed to pinpoint the roles of human cell proteins in HIV-1 integration. Other avenues of research include detailed characterization of preintegration complexes isolated from infected cells. Since each infected cell contains on average just one or two complexes, their detailed characterization has proven difficult. Novel biochemical techniques were proposed to surmount this obstacle, and additional experiments were proposed to construct preintegration complexes from scratch using purified protein and DNA components. The results of these experiments will significantly advance basic knowledge of how HIV-1 accomplishes its integration in human cells, which will contribute to the pharmacological goal of developing drugs that effectively inhibit virus integration during the development of AIDS in humans.

描述（由申请人提供）：人类免疫缺陷病毒1型（HIV-1）的复制导致人类艾滋病，制药行业开发的抗病毒药物是目前对抗HIV/艾滋病的最有效方法。HIV-1自身产生三种酶，逆转录酶、蛋白酶和整合酶，这三种酶对其生长至关重要。目前临床上的药物靶向逆转录酶和蛋白酶，因此整合酶是目前临床医生唯一不靶向的病毒酶。为了更多地了解整合酶的作用机制，本申请提出了在人类细胞感染期间，该酶如何发挥功能以整合其cDNA底物。在感染过程中，病毒形成由整合酶、cDNA和其他病毒以及人细胞蛋白组成的功能性整合前复合物。许多不同的人类蛋白质与HIV-1整合前复合物的功能有关，但尚不清楚在感染过程中需要这些蛋白质的程度。将进行各种蛋白质生物化学和病毒遗传实验，以确定人类细胞蛋白在HIV-1整合中的作用。其他研究途径包括从受感染细胞分离的整合前复合物的详细表征。由于每个受感染的细胞平均只含有一个或两个复合物，因此很难对其进行详细的表征。新的生物化学技术被提出来克服这一障碍，并提出了额外的实验，从头开始使用纯化的蛋白质和DNA成分构建整合前复合物。这些实验的结果将大大推进HIV-1如何在人体细胞中完成整合的基本知识，这将有助于开发在人类艾滋病发展过程中有效抑制病毒整合的药物的药理学目标。