HIV 1 INTEGRATION

描述：(改编自申请者摘要)本项目的目标 是为了检查整合反应，在这种反应中，人类免疫缺陷 病毒(HIV)DNA被整合到细胞DNA中，为了理解 整合在艾滋病发病机制中的作用。初步实验 建议用来表征HIV感染者血细胞中的整合 个人。将进行聚合酶链式反应(PCR)以检测 整合和未整合的HIV DNA水平，划分 HIV DNA在细胞质和核酸酶之间，以及未整合的形式 艾滋病毒DNA。调查人员预计，这些数据将使 任何观察到的低效的整合途径中的可能位置 整合。细胞类型对整合的影响也将是 检查过了。本应用程序的其余部分描述了一个组合 关注HIV整合酶(IN)的遗传和生化方法。 在3‘端特定位置含有突变的病毒粒子的感染 HIV Poll(指定病毒逆转录酶)基因的区域将是 检查集成中的特定缺陷。定量分析将 指出生产性艾滋病毒感染是否需要整合。至 开始定义IN的结构域，突变的IN蛋白将与 野生型(Wt)蛋白在检测系统中的活性 整合途径中的一步，包括DNA结合、内切酶、 共价连接和DNA连接。这些化验还将用于 不同致病毒株纯化整合酶的比较 潜力。这一计划的独特之处在于表达和 从Visna病毒中提纯，这是一种能够产生 没有整合的感染。五月份对Visna病毒的分析提供了 对HIV的洞察，其中可能催化效率相对较低 整合。

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.