HIV 1 INTEGRATION

描述：（改编自申请人的摘要）本项目的目标 是检查整合反应，其中人类免疫缺陷 病毒（HIV）DNA被整合到细胞DNA中，为了了解 整合在艾滋病发病机制中的作用。 初始实验 提出的是表征艾滋病毒感染者血细胞中的整合 个体 将进行聚合酶链反应（PCR）以测量 整合和未整合的HIV DNA水平， 细胞质和核酸酶之间的HIV DNA的，以及未整合的形式， HIV DNA 研究人员预计，这些数据将定位 可能的网站在整合途径中的任何观察到的效率低下， 一体化 细胞类型对整合的影响也将是 考察 本申请的其余部分描述了组合的 基因和生物化学的方法，重点是艾滋病毒整合酶（IN）。 病毒体在3 '端的确定位置含有突变的感染 HIV pol（指定病毒逆转录酶）基因的区域将被 检查整合中的具体缺陷。 定量分析将 说明是否需要融合才能感染艾滋病毒。 到 开始定义IN的结构域，突变的IN蛋白将与 野生型（wt）蛋白质在探索每种蛋白质的测定系统中的活性 整合途径中的步骤，包括DNA结合，核酸内切酶， 共价键和DNA连接。 这些试验还将用于 比较来自不同致病性病毒的纯化整合酶 潜力 该计划的独特之处在于表达和 从visna病毒纯化IN，visna病毒是一种能够生产 没有融合的感染。 对维斯纳病毒IN的分析可能提供 对HIV IN的深入了解， 一体化

DESCRIPTION: (Adapted from applicant's abstract) The goals of this project are to examine the integration reaction, in which human immunodeficiency virus (HIV) DNA is incorporated into cellular DNA, and to understand the role of integration in the pathogenesis of AIDS. Initial experiments proposed are to characterize integration in blood cells of HIV-infected individuals. Polymerase chain reaction (PCR) will be performed to measure the levels of integrated and unintegrated HIV DNA, the compartmentalization of HIV DNA between cytoplasm and nuclease, and the forms of unintegrated HIV DNA. The investigator anticipates that these data will localize the probable site in the integration pathway of any observed inefficiency of integration. The influence of cell type on integration will also be examined. The remaining sections of this application describes a combined genetic and biochemical approach that focuses on HIV integrase (IN). Infections with virions containing mutations at defined positions in the 3' region of the HIV pol (specifies viral reverse transcriptase) gene will be examined for specific defects in integration. Quantitative analysis will indicate whether integration is required for productive HIV infection. To begin to define domains of IN, mutated IN proteins will be compared with wild-type (wt) proteins for activity in assay systems that explore each step in the integration pathway, including DNA binding, endonuclease, covalent linkage and DNA joining. These assays will also be used to compare purified integrases from viruses with different pathogenic potential. The unique aspect of this plan involves expressing and purifying IN from visna virus, a lentivirus that is capable of productive infection without integration. Analysis of visna virus IN may provide insights into HIV IN which may be relatively inefficient at catalyzing integration.