A GENETIC ANALYSIS OF HIV-1 INTEGRATION

描述(改编自申请人的摘要)：本申请概述 进一步探索HIV-1整合和整合酶过程的研究 功能。申请者已经开发了一套82项收费的天气预报集 整合酶的簇丙氨酸突变体覆盖93%的带电残基， 并将这些插入到具有感染性的HIV-1分子克隆中。他提议 在3个特定目标中检查整合酶生物学的几个方面。目标1将 利用一组5个条件整合酶突变体来恢复内部和 抑制原始突变体的基因外病毒抑制突变体 表型。希望这将导致对病毒配体的识别 与整合酶蛋白进行关键的分子间接触 病毒复制。一种HIV穿梭载体系统已经开发出来，它可以 将环状DNA形式直接克隆到细菌中，这将有助于 这些抑制子突变体的回收和鉴定。特定目标2将 表征从环状DNA衍生出来的有限的基因表达 来自某些复制缺陷整合酶突变体。运动学，拷贝数， 并将使用实时荧光聚合酶链式反应来评估原代细胞中的蛋白质表达 以及记者的分析。这些研究可能与基因治疗方案有关 和疫苗的开发。目标3将进一步分析一种新发现的有效成分 核定位信号存在于整合酶的羧基末端。 该区域的突变将被用来评估该NLS在 未分裂细胞的感染。

DESCRIPTION (adapted from applicant's abstract): This application outlines studies to further explore the process of HIV-1 integration and integrase function. The Applicant has developed a synoptic set of 82 charged cluster-to-alanine mutants of integrase covering 93% of the charged residues, and inserted these into an infectious molecular clone of HIV-1. He proposes to examine several aspects of integrase biology in 3 Specific Aims. Aim 1 will utilize a panel of 5 conditional integrase mutants to recover intra- and extragenic viral suppressor mutants that suppress the original mutant phenotype. It is hoped that this will lead to identification of viral ligands that make critical intermolecular contact with the integrase protein during virus replication. An HIV shuttle vector system has been developed which allows direct cloning of circular DNA forms into bacteria, and which will facilitate recovery and characterization of these suppressor mutants. Specific Aim 2 will characterize the limited gene expression seen from circular DNA forms derived from certain replication-defective integrase mutants. Kinetics, copy number, and protein expression in primary cells will be assessed using real-time PCR and reporter assays. These studies may have relevance to gene therapy protocols and vaccine development. Aim 3 will further analyze a newly identified potent nuclear localization signal present in the carboxy terminus of integrase. Mutations in this region will be used to assess the role of this NLS in infection of non-dividing cells.