MOLECULAR MECHANISMS OF HIV 1 DNA INTEGRATION

HIV 1 DNA 整合的分子机制

• 批准号: 2443209
• 负责人: Samson A Chow
• 金额: $ 18.48万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2443209
• 关键词: DNA binding protein; cell free system; chimeric proteins; chromosome aberrations; feline immunodeficiency virus; human immunodeficiency virus 1; integrase; molecular site; nucleic acid structure; nucleoproteins; polymerase chain reaction; protein engineering; tissue /cell culture; virus DNA; virus infection mechanism; virus integration

An essential step during the life cycle of all retroviruses, including human immunodeficiency virus (HIV), is integration of a double-stranded DNA copy of the viral genome into a chromosome of the host cell. Two factors are critical for the integration process: the viral protein, integrase, and sequences at the ends of the linear viral DNA. Integrase removes two nucleotides from the 3' ends of a linear viral DNA molecule, and subsequently mediates a coupled cleavage-ligation reaction during which a staggered cut is made in the target DNA, and the resulting 5' ends of the target DNA are covalently joined to the recessed 3' ends of the viral DNA. The timing of the joining of the viral 5' ends to target DNA is presently not known, and the protein factors involved remain to be characterized. During integration, many sites in the host chromosome can be used as targets, although a wide variation in integration efficiency is observed among the target sites. The mechanism that determines target site specificity is not well understood. Since integration is required for retroviral replication and there is no recognized counterpart to integrase in normal cellular function, integration is an appealing target for developing specific inhibitors against retroviruses. The broad, long-term objective of this proposal is to further the understanding of the mechanism of HIV integration. The specific aims are (A) to examine target site selection during retroviral DNA integration, and (B) to study the kinetics and mechanisms of joining of the viral 5' end to target DNA, a poorly characterized final step of integration. The experimental design and methods for achieving these goals arc. (1) to construct chimeric proteins between HIV-1 and FIV integrases for determining the protein domain involved in selecting DNA sites for integration, and to use a PCR-based selection and amplification protocol for determining the DNA sequence optimal for integrase recognition, (2) to explore site-directed integration by studying in vitro and in vivo the activities and integration patterns of fusion proteins consisting of integraSe and a sequence-specific DNA binding protein, and (3) to use a novel strategy to study the timing of viral 3'- and 5'-end joining, and to develop an in vitro system for characterizing the 5'-end joining step and the factors required. The study may reveal potential novel targets for inhibitors and provide useful assays for drug screening. Information obtained from studying integration site selection may lead to a new approach for inserting exogenous genes at specific sites.

在所有逆转录病毒的生命周期中的重要步骤，包括 人免疫缺陷病毒（HIV）是双链的整合 病毒基因组的DNA拷贝成宿主细胞的染色体。二 因素对于整合过程至关重要：病毒蛋白， 整合酶和线性病毒DNA末端的序列。整合酶 从线性病毒DNA分子的3'末端去除两个核苷酸， 随后介导了耦合的裂解连接反应 在目标DNA中进行了交错的切割，由此产生的5' 目标DNA的末端共价连接到嵌入式的3'末端 病毒DNA。病毒5'结束的连接时间到目标的时间 目前尚不清楚DNA，涉及的蛋白质因子仍然存在 被描述。在整合过程中，主机染色体中的许多地点 可以用作目标，尽管集成的变化很大 在目标部位之间观察到效率。该机制 确定目标位点特异性尚不清楚。自从 逆转录病毒复制需要集成，没有 公认在正常细胞功能中的集成酶的对应物， 集成是开发特定抑制剂的吸引力的目标 反对逆转录病毒。该提议的广泛，长期目标是 进一步理解HIV整合机制。这 具体目的是（a）检查逆转录病毒期间的目标部位选择 DNA整合，（b）研究加入的动力学和机制 靶DNA的病毒5'结束，这是一个不当表征的最后一步 一体化。实现这些的实验设计和方法 目标弧。 （1）在HIV-1和FIV之间构建嵌合蛋白 积分用于确定选择DNA的蛋白质结构域 集成站点，并使用基于PCR的选择和扩增 确定集成酶最佳DNA序列的协议 识别，（2）通过研究中的地点指导的集成 体外和体内融合的活动和整合模式 由整合酶和序列特异性DNA结合组成的蛋白质 蛋白质和（3）使用一种新型策略来研究病毒3'-的时间 和5'-end加入，并开发一个体外系统来表征 5'端的加入步骤和所需的因素。该研究可能揭示 潜在的抑制剂新目标，并为药物提供有用的测定 筛选。从研究集成站点选择获得的信息 可能会导致一种新的方法，用于将外源基因插入特异性 站点。