ANALYSIS OF INTEGRASE IN REVERSE TRANSCRIPTION

逆转录整合酶分析

• 批准号: 6147643
• 负责人: JOHN Christopher KAPPES
• 金额: $ 24.43万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6147643
• 关键词: enzyme activity; genetic transcription; human immunodeficiency virus 1; integrase; molecular cloning; nucleic acid biosynthesis; nucleoproteins; polymerase chain reaction; protein protein interaction; protein sequence; recombinant proteins; virus infection mechanism; virus integration

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.

我们对 HIV-1 病毒生命周期早期事件的理解仍然存在很大差距。 这部分是由于 HIV-1 蛋白编码和组装成病毒颗粒的方式所致。 逆转录病毒酶在 Gag-Pol 前体多蛋白的 C 端 Pol 组分内编码。 所有逆转录病毒至少编码酶活性：蛋白酶（PR）、聚合酶和核糖核酸酶H（RNase H）活性、逆转录酶（RT）和整合酶（IN）。 HIV-1 编码并合并 PR、RT 和 IN 作为 160 kDA Gag-Pol 前体 (Pr160Gag/Pol) 的 Pol 成分。 Gag-Pol 内的突变是多效性的，因为它们可以在组装和成熟（后期）过程中影响前体蛋白，以及成熟 Gag（MA、CA、NC、p6）和 Pol（PR、RT、IN）的功能）蛋白质，经过蛋白水解加工和病毒感染（早期事件）。例如，In 突变已被证明可以通过各种机制并在病毒生命周期的不同阶段改变病毒复制。 这可能解释了已报道的IN突变病毒的多种表型，包括那些在组装、病毒粒子形态、逆转录、核输入和原病毒整合方面存在缺陷的病毒。 最近，我们通过反式表达作为 Vpr-IN 融合蛋白，分析了 HIV-1 IN 蛋白的功能依赖于其作为 Gag-Pol 一部分的表达。 我们的分析表明，成熟的 IN 蛋白本身是体内有效逆转录所必需的。 我们的数据和其他数据还表明，DNA 合成缺陷发生在起始和延伸之前或之前，因为 IN 突变体合成等量的早期和晚期 DNA，尽管明显少于野生型病毒。 这些发现表明，我们第一次可以在复制病毒（体内）的背景下，将早期和晚期事件中的 IN 分析分开。该应用的中心假设是，IN 蛋白通过与受感染细胞内逆转录复合物的其他病毒成分相互作用，在逆转录起始阶段发挥重要作用。需要进一步分析来了解 IN 增强病毒 DNA 合成的机制。 该提案的具体目标是：（1）定义病毒生命周期中IN促进RT的阶段； (2) 定义影响 IN 增强 RT 能力的特定病毒成分； (3) 确定有效逆转录酶是否需要其他逆转录病毒IN蛋白； (4) 绘制 DNA 合成所需的 IN 蛋白的决定簇。 这些研究将有助于确定 IN 蛋白在核蛋白复合物背景下的 HIV-1 RT 中的作用，并了解 IN 蛋白与核蛋白复合物的其他成分之间的特定分子相互作用，这些相互作用是在受感染细胞中进行有效 RT 所需的。