Biochemical Mechanism of HIV DNA Integration

描述(申请人提供)：复制人类免疫缺陷病毒1型(HIV-1)会导致人类感染艾滋病，制药业开发的抗病毒药物是目前对抗艾滋病毒/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。目前临床上的药物以逆转录酶和蛋白酶为靶点，因此整合酶是目前唯一未被临床医生靶向的病毒酶。为了更多地了解整合酶的作用机制，该应用程序建议破译在人类细胞感染过程中，该酶如何作用于整合其cdna底物。在感染过程中，病毒形成一个功能前整合复合体，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前整合复合体的功能有关，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学实验，以确定人类细胞蛋白质在HIV-1整合中的作用。其他研究途径包括从受感染细胞中分离出来的整合前复合体的详细特征。由于每个受感染的细胞平均只含有一个或两个复合体，它们的详细描述被证明是困难的。为了克服这一障碍，人们提出了新的生化技术，并提出了更多的实验，以利用纯化的蛋白质和DNA组分从头开始构建预整合复合体。这些实验的结果将大大提高关于HIV-1如何在人类细胞中实现整合的基础知识，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。

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.