ANALYSIS OF INTEGRASE IN REVERSE TRANSCRIPTION

在我们对HIV-1病毒生命周期早期事件的理解方面仍然存在很大差距。 这部分是由于HIV-1蛋白编码和组装到病毒颗粒中的方式。 逆转录病毒酶在Gag-Pol前体多蛋白的C末端Pol组分内编码。 所有逆转录病毒至少编码酶活性：逆转录酶（RT）的蛋白酶（PR）、聚合酶和核糖核酸酶H（RNase H）活性以及整合酶（IN）。 HIV-1编码并整合PR、RT和IN作为160 kDA Gag-Pol前体（Pr 160 Gag/Pol）的Pol组分。 Gag-Pol内的突变是多效性的，因为它们可以在组装和成熟（晚期）期间影响前体蛋白，以及在蛋白水解加工和病毒感染（早期事件）之后影响成熟Gag（MA、CA、NC、p6）和Pol（PR、RT、IN）蛋白的功能。例如，In中的突变已显示通过各种机制和在病毒生命周期的不同阶段改变病毒复制。 这可能解释了IN突变病毒的不同表型，包括在装配、病毒体形态、逆转录、核输入和整合theprovirus方面存在缺陷的病毒。 最近，我们分析了HIV-1 IN蛋白的功能依赖于其作为Gag-Pol的一部分的表达，通过作为Vpr-IN融合蛋白的反式表达。 我们的分析表明，成熟的IN蛋白本身是需要在体内进行有效的逆转录。 我们的数据和其他人也表明，DNA合成缺陷是在或之前的启动和延长，因为IN突变体合成等量的早期和晚期DNA，虽然显着低于野生型病毒。 这些研究结果表明，我们可以，第一次，解偶联分析IN在早期和晚期事件中，在复制病毒的情况下（体内）。本申请的中心假设是IN蛋白通过与感染细胞内逆转录复合物的其他病毒组分相互作用在逆转录的起始阶段起重要作用。进一步的分析是必要的，以了解IN增强病毒DNA合成的机制。 该提案的具体目标是：（1）确定IN促进RT的病毒生命周期阶段;（2）确定影响IN增强RT能力的特定病毒组分;（3）确定有效RT是否需要其他逆转录病毒IN蛋白;（4）绘制DNA合成所需的IN蛋白决定簇。 这些研究将有助于定义IN蛋白在HIV-1 RT中的作用，并了解IN蛋白与受感染细胞中有效RT所需的核蛋白复合物的其他组分之间的特异性分子相互作用。

There remains a large gap in our understanding of the early events of the HIV-1 virus life cycle. This is due in part to the manner in which HIV-1 proteins are encoded and assembled into the virus particle. The retroviral enzymes are encoded within the C- terminal Pol component of the Gag-Pol precursor polyprotein. All retroviruses encode at least for enzyme activities: protease (PR), polymerase and ribonuclease H (RNase H) activities of the reverse transcriptase (RT), and integrase (IN). HIV-1 encodes and incorporates PR, RT and IN as the Pol components of a 160 kDA Gag-Pol precursor (Pr160Gag/Pol). Mutations within Gag-Pol are pleiotropic, since they can affect the precursor protein during assembly and maturation (late stage), as well as the functions of the mature Gag (MA, CA, NC, p6) and Pol (PR, RT, IN) proteins, after proteolytic processing and virus infection (early events). For example, mutations in In have been shown to alter virus replication through various mechanisms and at different stages in the virus life cycle. This likely explains the diverse phenotypes that have been reported for IN mutant viruses, including those with defects in assembly, virion morphology, reverse transcription, nuclear import, and integration of theprovirus. Recently, we analyzed the function of the HIV-1 IN protein dependently of its expression as part of Gag-Pol, via expression in trans as a Vpr-IN fusion protein. Our analysis has revealed that the mature IN protein itself is required for efficient reverse transcription in vivo. Our data and others also indicate that the DNA synthesis defect is at or prior to initiation and elongation, since the IN mutants synthesize an equal amount of early and late DNA, albeit significantly less than that of wild-type virus. These findings demonstrated that we can, for the first time, uncouple the analysis of IN in early and late events, in the context of a replicating virus (in vivo). The central hypothesis of this application is that the IN protein plays an important role in the initiation stage of reverse transcription through interactions with other viral components of the reverse transcription complex within the infected cell. Further analysis is necessary to understand the mechanisms by which IN augments viral DNA synthesis. The specific objectives of this proposal are: (1) to define the stage in the virus life cycle that IN promotes RT; (2) to define specific viral components that affect the ability of IN to augment RT; (3) to determine whether other retroviral IN proteins are required for efficient RT; and (4) to map the determinants of the IN protein that are required for DNA synthesis. These studies will help to define the IN protein's role in HIV-1 RT in the context of the nucleoprotein complex, and understand the specific molecular interactions between the IN protein and other components of the nucleoprotein complex that required for efficient RT in the infected cell.