TRANSCOMPLEMENTATION SYSTEM TO STUDY PROVIRUS SYNTHESIS

像所有逆转录病毒一样，艾滋病毒有必要将其 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。这个 HIV-I RT蛋白具有多种功能，需要相互作用 与病毒粒子内的几种病毒和细胞蛋白以及 受感染的宿主细胞是完全合成 病毒DNA。在大多数RT研究中，细菌表达的RT蛋白具有 用于二级结构分析，以确定 子功能域并帮助理解结构-功能 两性关系。这些努力的结果做出了贡献 对目前对反转录的理解有很大的帮助 体外实验，在分子水平上。最近的研究表明， 在被感染的细胞中(体内)转录比 是因为感染细胞中的前病毒DNA合成 需要其他病毒和细胞蛋白。目前还没有 可用于体内反转录研究的良好系统 部分，因为操作RT的固有困难 表达和掺入不依赖于GAG/POL。我们最近 对HIV辅助蛋白的研究表明，它们可以被开发 作为将功能蛋白整合到病毒粒子中的载体，通过 异源融合分子在反式中的表达。我们已经展示了 蛋白水解酶(PR-突变体)整合酶(IN)，并逆转 转录酶(RT)可以通过表达被掺入病毒粒子中 在反式VPR融合蛋白中。重要的是，我们的初步数据 证明IN和RT的DNA合成存在缺陷 用VPR-RT和VPR-RT反式互补可恢复前病毒 VPR-IN融合蛋白。在此应用程序中，我们 提议利用VPR(可能还有GAG)作为一种工具来 在病毒粒子中掺入功能性RT蛋白建立模型 有助于研究逆转录病毒过程的系统 被感染细胞中的逆转录。的中心假说 这个项目是通过定义必要的关键组件 RT函数的高效反式互补，我们可以开发一种 一种研究反转录过程的模型系统 与生物学相关的背景。在申请中，我们建议 构建RT缺陷主干前病毒，它可以 通过反式互补有效挽救(DNA合成) 带有RT融合蛋白，其功能与病毒相似- 编码RT。为了实现这些目标，我们建议：i.确定 RT编码序列在Gag/Pol表达中的作用，以及 GAG蛋白的蛋白水解性加工；2.分析 前病毒编码的RT突变体对细胞功能的影响 VPR-RT融合蛋白的表达；3.分析RT的作用 逆转录酶缺陷HIV-I反式互补的融合伙伴； 反式互补分析RT缺陷病毒复制 使用稳定的细胞系；以及5.研究IN的作用 被感染细胞中的逆转录。HIV-I逆转的研究 感染细胞中的转录将有助于理解准确的 前病毒DNA合成和易化的分子机制 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.