ANALYSIS OF INTEGRASE IN REVERSE TRANSCRIPTION

我们对艾滋病毒-1病毒生命周期早期事件的理解仍然存在很大差距。这在一定程度上是由于HIV-1蛋白被编码并组装到病毒颗粒中的方式。逆转录病毒酶编码在Gag-Pol前体多蛋白的C-末端Pol成分中。所有的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)活性的逆转录酶(RT)和整合酶(IN)。HIV-1编码PR、RT和IN作为160KDA Gag-Pol前体(Pr160Gag/Pol)的Pol组分。Gag-Pol内的突变是多效性的，因为它们可以影响装配和成熟过程中的前体蛋白(后期)，以及成熟的Gag(MA、CA、NC、p6)和Pol(PR、RT、IN)蛋白的功能，在蛋白分解处理和病毒感染(早期事件)之后。例如，在病毒生命周期的不同阶段，in基因的突变已被证明通过各种机制改变病毒复制。这可能解释了已报道的IN突变病毒的不同表型，包括那些在组装、病毒粒子形态、反转录、核输入和前病毒整合方面存在缺陷的病毒。最近，我们通过反式表达VPR-IN融合蛋白，分析了HIV-1IN蛋白的功能依赖于其作为Gag-Pol的一部分的表达。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也表明，DNA合成缺陷处于或早于起始和延伸，因为IN突变体合成相同数量的早期和晚期DNA，尽管明显低于野生型病毒。这些发现表明，我们第一次可以在复制病毒(体内)的背景下，分离早期和晚期事件中IN的分析。这一应用的中心假设是，IN蛋白通过与感染细胞内反转录复合体的其他病毒成分相互作用，在反转录的起始阶段发挥重要作用。为了了解IN促进病毒DNA合成的机制，有必要进行进一步的分析。这项建议的具体目标是：(1)确定IN促进RT的病毒生命周期阶段；(2)定义影响IN增强RT能力的特定病毒成分；(3)确定有效RT是否需要其他逆转录病毒IN蛋白；以及(4)绘制DNA合成所需IN蛋白的决定因素图。这些研究将有助于在核蛋白复合体的背景下确定IN蛋白在HIV-1RT中的作用，并了解IN蛋白与感染细胞中有效RT所需的核蛋白复合体其他成分之间的特定分子相互作用。

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.