A GENETIC ANALYSIS OF HIV-1 INTEGRATION

描述（改编自申请人摘要）：本申请概述 进一步探索HIV-1整合过程和整合酶的研究 功能申请人已经开发了一套82个带电的 整合酶的簇-丙氨酸突变体覆盖93%的带电残基， 并将其插入HIV-1的感染性分子克隆中。他拟 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细胞内和 抑制原始突变体的基因外病毒抑制突变体 表型。希望这将导致病毒配体的鉴定 与整合酶蛋白进行关键的分子间接触， 病毒复制已经开发了一种HIV穿梭载体系统， 将环状DNA直接克隆到细菌中，这将有助于 这些抑制突变体的回收和表征。具体目标2将 描述了从来源于DNA的环状DNA形式中看到的有限基因表达， 来自某些复制缺陷型整合酶突变体。动力学，拷贝数， 使用实时PCR评估原代细胞中的蛋白质表达 和报告基因测定。这些研究可能与基因治疗方案有关 和疫苗开发。Aim 3将进一步分析一种新发现的强效 整合酶羧基端的核定位信号。 该区域的突变将用于评估该NLS在以下方面的作用： 感染非分裂细胞。

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.