TRANSCOMPLEMENTATION SYSTEM TO STUDY PROVIRUS SYNTHESIS

像所有逆转录病毒一样，艾滋病毒需要将其 将基因组RNA转化为双链DNA。这个逆转的过程 转录由逆转录酶（RT）催化。的 HIV-I RT蛋白具有多种功能，需要相互作用 在病毒粒子内有几种病毒和细胞蛋白， 被感染的宿主细胞，其是完全合成 病毒DNA在大多数RT研究中，细菌表达的RT蛋白 用于二级结构分析，以识别 亚功能结构域，并帮助理解结构-功能 关系。这些努力的结果有助于 这对目前对逆转录的理解有很大的帮助， 在体外，在分子水平上。最近的研究表明， 受感染细胞中的转录（体内）比 这是预料之中的，因为在感染细胞中的前病毒DNA合成 需要其他病毒和细胞蛋白质。目前没有 良好的系统可用于研究体内逆转录， 部分原因是由于操纵RT的固有困难 表达和掺入独立的gag/pol。我们最近的 对HIV辅助蛋白的研究表明， 作为将功能性蛋白质整合到病毒体中的载体， 作为异源融合分子的反式表达。我们已经表明 蛋白酶（PR突变体）整合酶（IN），并逆转 转录酶（RT）可通过表达掺入病毒体中 以Vpr-融合蛋白的形式反式表达。重要的是，我们的初步数据 证明IN和RT缺陷的DNA合成 前病毒可以通过与Vpr-RT的反式互补而恢复， Vpr-IN融合蛋白。在本申请中，我们 建议利用Vpr（可能还有Gag）作为工具， 将功能性RT蛋白掺入病毒体中以建立模型 该系统将有助于研究逆转录病毒的过程， 在感染细胞中的逆转录。的中心假设 这个项目是，通过定义必要的关键组成部分， RT功能的有效反式互补，我们可以开发一种 研究逆转录过程的模型系统 生物学相关背景。在申请中，我们建议 构建RT缺陷型骨架前病毒，其可以 通过反式互补有效地挽救（DNA合成） 一种RT融合蛋白，其功能模拟病毒的功能， 为了实现这些目标，我们提出：i.确定 RT编码序列在Gag/Pol表达中的作用，以及 Gag蛋白的蛋白水解加工; 2.分析 由原病毒编码的RT突变体对 Vpr-RT融合蛋白; 3.分析RT的效果 融合伴侣对RT缺陷型HIV-I的反式互补作用; 4. 通过反式互补分析RT缺陷型病毒复制 使用稳定的细胞系;和5.研究IN的作用 在感染细胞中的逆转录。HIV-1逆转的研究 在感染细胞中的转录将有助于理解 前病毒DNA合成和促进的分子机制 开发新的抗逆转录病毒策略。

Like all retroviruses, it is necessary for HIV to convert its genomic RNA into double stranded DNA. This process of reverse transcription is catalyzed by the reverse transcriptase (RT). The HIV-I RT protein has multiple functions and requires interaction with several viral and cellular proteins within the virion and the infected host cell that are necessary for complete synthesis of the viral DNA. In most RT studies bacterially expressed RT protein has been used for secondary structure analysis, to identify subfunctional domains and to help understand structure-function relationships. The results of these efforts have contributed greatly to the current understanding of reverse transcription in vitro, at the molecular level. Recent studies suggest that reverse transcription in the infected cell (in vivo) is more complex than was expected because proviral DNA synthesis in infected cells requires other viral and cellular proteins. Currently there are not good systems available to study reverse transcription in vivo, in part, because of inherent difficulties in manipulating RT expression and incorporation independent of gag/pol. Our recent studies on HIV accessory proteins have shown they can be exploited as vehicles to incorporate functional proteins into virions by expression in trans as heterologous fusion molecules. We have shown that the protease (PR-mutant) integrase (IN), and reverse transcriptase (RT) can be incorporated into virions by expression in trans as Vpr-fusion proteins. Importantly, our preliminary data demonstrate that the DNA synthesis of IN and RT defective proviruses can be restored by trans complementation with Vpr-RT and Vpr-IN fusion proteins, respectively. In this application, we proposes to exploit Vpr (and possibly Gag) as a vehicle to incorporate functional RT protein into virions to establish a model system that will facilitate studies on the processes of retroviral reverse transcription in infected cells. The central hypothesis of this project is that, by defining the key components necessary for efficient trans complementation of RT function, we can develop a model system to study processes of reverse transcription in a biologic relevant context. In the application, we propose to construct an RT defective backbone provirus, which can be effectively rescued (for DNA synthesis) by trans complementation with an RT fusion protein whose function mimics that of the virus- encoded RT. To achieve these goals we propose: i. To determine the role of the RT coding sequence in Gag/Pol expression, and proteolytic processing of the Gag protein; 2. To analyze the effects of RT mutants encoded by the provirus on the function of the Vpr-RT fusion protein; 3. To analyze the effect of the RT fusion partner on trans complementation of RT defective HIV-I; 4. To analyze RT defective virus replication by trans complementation using stable cell lines; and 5. To investigate the role of IN reverse transcription in infected cells. Study of HIV-I reverse transcription in infected cell will help to understand the precise molecular mechanisms of provirus DNA synthesis and facilitate development of novel antiretroviral strategies.