ANALYSIS OF INTEGRASE IN REVERSE TRANSCRIPTION

逆转录整合酶分析

基本信息

批准号：
6632295
负责人：
JOHN Christopher KAPPES
金额：
$ 25.11万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-06-01 至 2006-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6632295
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

项目摘要

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病毒生命周期的早期事件的理解仍然存在很大的差距。这部分是由于将HIV-1蛋白编码并组装到病毒颗粒中的方式。逆转录病毒酶被编码在GAG-POL前体多蛋白的C-末端POL成分中。所有逆转录病毒至少用于酶活性：逆转录酶（RT）（RT）和综合酶（IN）的蛋白酶（PR），聚合酶和核糖酶H（RNase H）活性。 HIV-1编码并掺入PR，RT，并作为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期间（晚期）以及成熟GAG（MA，CA，CA，NC，P6）和POL（PR，RT，IN）蛋白质，蛋白水解处理和病毒感染（早期事件）的功能（MA，CA，NC，RT，IN）。例如，已证明IN突变通过各种机制和病毒生命周期的不同阶段改变病毒复制。这可能解释了突变病毒中报告的多种表型，包括那些在组装，病毒式形态，逆转录，核进口和促性病毒整合的缺陷的表型。最近，我们通过在反式中作为VPR-IN融合蛋白的表达在蛋白质中分析了HIV-1在蛋白质中的功能。我们的分析表明，蛋白质本身的成熟是在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的合成缺陷处于启动和伸长之前或伸长率之前，因为突变体合成相等量的早期和晚DNA，尽管大大低于野生型病毒的DNA。这些发现表明，在复制病毒（体内）的背景下，我们可以第一次解开对早期和晚期事件的分析。该应用的中心假设是，蛋白质中的蛋白质在逆转录的起始阶段通过与感染细胞内反转录复合物的其他病毒成分相互作用而起着重要作用。需要进一步分析以了解增强病毒DNA合成的机制。该提案的具体目标是：（1）定义促进RT中病毒生命周期的阶段；（2）定义影响IN增强RT能力的特定病毒成分；（3）确定有效RT是否需要其他蛋白质中的其他逆转录病毒；（4）绘制DNA合成所需的蛋白质中的决定因素。这些研究将有助于在核蛋白复合物的背景下定义蛋白质在HIV-1 RT中的作用，并了解在受感染细胞中有效RT所需的蛋白质与核蛋白复合物的其他成分之间的特定分子相互作用。