GENETIC ANALYSES OF THE HIV 1 INITIATION COMPLEX

反转录的起始发生在引物结合部位 (PBS)，其与3‘端18结果核苷酸互补 3.我们实验室的研究有恶魔- 研究表明，U5和PBS内的突变导致病毒 稳定地利用tRNAHis或tRNAMet启动逆转录。 基于这些研究，我们得出结论，rna基因组是一种主要的 选择用于启动逆转反应的tRNA的决定因素 抄写。描述相互作用的参数 在tRNALys、3和PBS之间，则代表着 治疗学。为了进一步定义U5-PBS与 TRNA，我们将使用遗传方法来识别关键区域 在选择tRNA所需的U5内。以下是具体的 提出目标：--具体目标1：确定 美国境内维持PBS与tRNAMet互补的三个地区 或者是tRNAHis。我们已经使用RNA折叠程序鉴定了三个RNA U5-PBS内的结构。在这些区域使用已定义的突变体， 我们将描述反转录/病毒的重要性 复制。平行实验将与相关的 使用感染原代细胞的病毒的突变体。具体目标2： 相关的是U5的突变破坏了与 用来启动逆转录的tRNA。一种独特的RT/PCR方法 将被用来分析逆转录的启动。一个 将使用互补系统来分析 U5中的突变对逆转录的完成有影响。 具体目标3：研究tRNA U5-PBS相互作用 发生在病毒释放之前。细胞核和细胞质提取液将 由野生型或突变型前病毒感染的细胞产生 基因组。将使用RT/PCR方法来确定是否正确 反转录的启动发生在添加的 病毒蛋白。我们的遗传方法与 化学/酶研究将提供RNA-RNA的清晰图景 U5-PBS-tRNA之间的相互作用。从造型和结构 分析，我们将完善我们对关键区域的理解 U5-PBS-tRNA相互作用。最后，我们独特的病毒系统 利用tRNAHis或tRNAMet启动逆转录将是 用于测试新疗法的特异性和有效性 旨在抑制野生型tRNA-U5-PBS的相互作用 基因组。

Initiation of reverse transcription occurs at the primer binding site (PBS), which is complementary to the 3' terminal 18 results nucleotides of the cellular tRNALys, 3. Studies from our laboratory have demon- strated that mutations within U5 and the PBS results in virus which stably utilizes tRNAHis or tRNAMet to initiate reverse transcription. Based on these studies, we conclude that the RNA genome is a major determinant for the selection of the tRNA used to initiate reverse transcription. Delineation of the parameters for the interaction between tRNALys,3 and the PBS then represents an ideal new target for therapeutics. To further define the interaction between U5-PBS and the tRNA, we will use a genetic approach to identify critical regions within U5 required for selection of the tRNA. The following specific aims are proposed: -Specific Aim 1: To establish the importance of three regions within US for maintenance of PBS complementary to tRNAMet or tRNAHis. We have used RNA fold programs to identify three RNA structures within the U5-PBS. Using defined mutants in these regions, we will delineate the importance in reverse transcription/viral replication. Parallel experiments will be performed with relevant mutants using viruses which infect primary cells. Specific Aim 2: To correlate that mutations in U5 disrupt the capacity to interact with the tRNA used to initiate reverse transcription. A unique RT/PCR method will be used to analyze initiation of reverse transcription. A complementation system will be used to analyze the effects that mutations in U5 have on the completion of reverse transcription. Specific Aim 3: To investigate whether the tRNA U5-PBS interaction occurs prior to release of virus. Nuclear and cytoplasmic extracts will be generated from cells transfected with wild type or mutant proviral genomes. The RT/PCR method will be used to determine if correct initiation of reverse transcription occurs in the presence of added viral proteins. The integration of our genetic approach with the chemical/enzymatic studies will provide a clear picture of the RNA-RNA interaction between U5-PBS-tRNA. From the modeling and structural analysis, we will refine our understanding of critical regions of the U5-PBS-tRNA interaction. Finally, our unique viral systems which utilize tRNAHis or tRNAMet to initiate reverse transcription will be used to test the specificity and effectiveness of new therapeutics designed to inhibit the tRNA-U5-PBS interaction of the wild type genome.