Biochemical Mechanism of HIV DNA Integration

描述（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，而制药行业开发的抗病毒药物是目前对抗艾滋病最有效的方法。 HIV-1 自身产生三种对其生长至关重要的酶：逆转录酶、蛋白酶和整合酶。目前临床上的药物主要针对逆转录酶和蛋白酶，因此整合酶是目前临床医生未针对的唯一病毒酶。为了更多地了解整合酶的作用机制，本申请旨在破译该酶在人类细胞感染过程中如何发挥整合其 cDNA 底物的作用。在感染过程中，病毒形成功能性预整合复合物，由整合酶、cDNA 和其他病毒以及人类细胞蛋白组成。许多不同的人类蛋白质与 HIV-1 预整合复合物的功能有关，但尚不清楚感染期间这些蛋白质的需要程度。将进行各种蛋白质生物化学和病毒遗传实验，以查明人类细胞蛋白质在 HIV-1 整合中的作用。其他研究途径包括从感染细胞中分离出的预整合复合物的详细表征。由于每个受感染的细胞平均只含有一两个复合物，因此对其详细表征已被证明是困难的。人们提出了新的生化技术来克服这一障碍，并提出了额外的实验来使用纯化的蛋白质和 DNA 成分从头开始构建预整合复合物。这些实验的结果将显着推进关于HIV-1如何在人类细胞中整合的基础知识，这将有助于实现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。

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.