IN VITRO STRUCTURAL ANALYSIS OF THE HIV 1 INITIATION COMPLEX

复制人类免疫缺陷病毒的关键一步 (HIV)是由HIV形成的一种引发复合体 基因组和细胞tRNA。这种二元RNA复合体与 病毒编码的蛋白质包括逆转录酶(RT)和 核衣壳蛋白(NCp7)，介导了引物tRNA的延长 从R-U5形成短DNA中间体(负链终止DNA) 病毒RNA的序列。选择性地使用细胞tRNALys，3 在这个HIV-1复制的早期阶段和两个互补阶段 逆转录病毒R-U5内的序列已被证明是重要的 在早期复制步骤中。病毒内的18个核苷酸序列 被称为引物结合位点(PBS)的基因组与3‘端是互补的 TRNALYS，3的末端和上游的一个富腺嘌呤序列(A-环) 与tRNALYS互补，3个反密码子。形成的组织 起始复合体需要展开两个病毒模板 和tRNA引物，形成新的RNA结构。而当 RT在HIV复制的这个初始阶段中的作用是广泛的- LY调查对结构要求知之甚少 启动复合体。该项目的目标将是提供 结构信息是首先制定一个可信的 引发络合物的三级结构模型和二级结构 通过关键基因的定点突变对该模型进行功能测试 结构元素。这项建议的具体目的是1) 确定tRNALys，3-HIV-1复制的二级结构 引发络合物和与其形成的交替引发络合物 TRNAHis和tRNAMet；2)建立选择性三级结构 分子内和分子间交联剂的限制 TRNALys、3-HIV-1起始物和交替起始物 由tRNAHis和tRNAMet组成；3)建立体外选择 寻找新的tRNA-HIV启动复合体的策略。这些研究 将提供对结构特征的首次详细分析 启动HIV-1逆转所需的RNA：RNA相互作用 抄写。艾滋病毒生命周期中的这一关键步骤是一个吸引人的 药物干预的目标和来自我们的 研究将为此类化合物的开发提供基础。

A critical step in the replication of human immunodeficiency virus (HIV) is the formation of an initiation complex composed of the HIV genome and a cellular tRNA. This binary RNA complex in conjunction with viral encoded proteins including, reverse transcriptase (RT) and the nucleocapsid protein (NCp7), mediates the elongation of the primer tRNA to form a short DNA intermediate (minus-strand stop DNA) from R-U5 sequences of the viral RNA. The cellular tRNALys, 3 is selectively used in this early stage of HIV-1 replication and two complementary sequences within the retrovirus R-U5 have been shown to be important in early replication steps. An 18 nucleotide sequence within the virus genome termed the primer binding site (PBS) is complementary to the 3' terminus of tRNALYS, 3 and an adenine rich sequence (A-loop) upstream of the PBS is complementary to tRNALYS, 3 anti-codon. Formation of the initiation complex requires the unfolding of both the viral template and tRNA primer and the formation of a new RNA structure. While the role of RT in this initial stage of HIV replication has been extensive- ly investigated little is known about the structural requirements for the initiation complex. The goal of this project will be to provide the structural information necessary to first formulate a plausible tertiary structure model of the initiation complex and second to functionally test this model by site directed mutagenesis of critical structural elements. The specific aims of this proposal are 1) to determine the secondary structure of the tRNALys, 3-HIV-1 replication initiation complex and alternative initiation complexes formed with tRNAHis and tRNAMet; 2) to establish selective tertiary structure constraints by intra- and inter- molecular crosslinking of the tRNALys,3-HIV-1 initiation complex and alternative initiation complexes formed with tRNAHis and tRNAMet; 3) to develop an in vitro selection strategy to identify novel tRNA-HIV priming complexes. These studies will provide the first detailed analysis of the structural features of the RNA:RNA interactions required for initiation of HIV-1 reverse transcription. This critical step in the HIV life-cycle is an appealing target for drug intervention and the structural information from our studies will provide the basis for the development of such compounds.