Biochemical Mechanism of HIV DNA Integration

描述（由申请人提供）：逆转录的人类免疫缺陷病毒1型（HIV-1）cDNA整合到感染细胞的染色体中对于病毒复制是必需的。因此，HIV-1整合酶（IN）酶和整合步骤是开发抗HIV/AIDS抗病毒药物的有吸引力的目标。了解HIV-1在体内整合的详细机制对于有效的抗IN和抗整合药物开发至关重要。体内整合由病毒感染和逆转录后形成的大核蛋白预整合复合物（PIC）介导，并且从感染细胞分离的PIC可以在体外将其内源性cDNA拷贝整合到添加的靶DNA中。父R 01赠款侧重于HIV-1 PIC的结构和功能。在这项提案中，两种不同的创新技术将被用于显着提高研究的速度和质量。最近描述的实时定量（RQ）-PCR测定定量水平的体内整合以及体外整合活性的PIC将驱动实验设计。RQ-PCR PIC整合试验将使检测体外整合的灵敏度比目前使用的Southern印迹试验提高约10，000倍，并将获得结果所需的时间从约一周缩短至一天。这将显着影响研究的能力，直接和快速地解决核心问题，包括确定宿主蛋白辅因子在HIV-1整合中的作用，以及体内整合所需的IN蛋白-蛋白和蛋白-DNA相互作用的关键细节。此外，小干扰RNA，一种以前无法获得的技术敲低哺乳动物细胞系中的基因表达，将与RQ-PCR一起使用，以确定以前涉及的宿主蛋白因子在体内HIV-1整合中的作用。结果将确定宿主因子在HIV-1整合中的作用，以及HIV-1体内整合所必需的PIC内的蛋白质-蛋白质和蛋白质-DNA相互作用。这些结果将大大增加对HIV-1整合的理解，因为它发生在受感染的细胞中，这反过来将有助于发现和开发针对HIV-1 IN和整合的抗病毒药物。

DESCRIPTION (provided by applicant): Integration of reverse transcribed human immunodeficiency virus type 1 (HIV-1) cDNA into a chromosome of an infected cell is essential for virus replication. Thus, the HIV-1 integrase (IN) enzyme and integration step are attractive targets for developing antiviral drugs against HIV/AIDS. Understanding the detailed mechanism of HIV-1 integration in vivo is critical for effective anti-IN and anti-integration drug development. Integration in vivo is mediated by large nucleoprotein preintegration complexes (PICs) that form following viral infection and reverse transcription, and PICs isolated from infected cells can integrate their endogenous cDNA copy into an added target DNA in vitro. The parent R01 grant focuses on the structure and function of HIV-1 PICs. In this proposal two different innovative technologies that were unavailable when the parent grant was written will be used to significantly enhance the pace and quality of research. Recently described real-time quantitative (RQ)-PCR assays for quantifying levels of in vivo integration as well as the in vitro integration activity of PICs will drive the experimental design. The RQ-PCR PIC integration assay will increase the sensitivity of detecting in vitro integration approximately 10,000-fold over the currently used Southern blotting assay, as well as reduce the time required for results from approximately one week to one day. This will significantly impact the ability of the research to move directly and rapidly toward addressing the central issues which include determining the roles of host protein cofactors in HIV-1 integration as well as critical details of IN protein-protein and protein-DNA interactions required for integration in vivo. Additionally, small-interfering RNA, a previously unavailable technique for knocking-down the expression of genes in mammalian cell lines, will be used alongside RQ-PCR to determine the roles of previously-implicated host protein factors in HIV-1 integration in vivo. The results will determine the roles of host factors in HIV-1 integration, as well as protein-protein and protein-DNA interactions within PICs essential for HIV-1 integration in vivo. These results will significantly add to the understanding of HIV-1 integration as it occurs in infected cells, which will in turn aid the discovery and development of antiviral drugs targeted against HIV-1 IN and integration.