MOLECULAR MECHANISMS OF HIV 1 DNA INTEGRATION

在所有逆转录病毒的生命周期中的一个重要步骤， 人类免疫缺陷病毒（HIV），是一种整合的双链 将病毒基因组的DNA拷贝到宿主细胞的染色体中。两 整合过程的关键因素是：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 ′端除去两个核苷酸， 并随后介导偶联的裂解-连接反应， 其中在靶DNA中进行交错切割，并且得到的5' 靶DNA的末端共价连接到靶DNA的凹进的3'末端， 病毒DNA病毒5'端连接至靶分子的时机 DNA目前还不清楚，涉及的蛋白质因子仍然是未知的。 被定性。在整合过程中，宿主染色体上的许多位点 可以用作目标，尽管集成的变化很大， 在靶位点中观察到效率。的机制 决定靶位点特异性的因素尚不清楚。以来 逆转录病毒复制需要整合， 在正常细胞功能中与整合酶对应， 整合是开发特异性抑制剂的吸引人的目标 对抗逆转录病毒这项建议的广泛和长期目标是 进一步了解艾滋病毒整合的机制。的 具体目标是（A）检查逆转录病毒过程中的靶位点选择 DNA整合，和（B）研究连接的动力学和机制 将病毒5'端的DNA连接到靶DNA，这是一个表征不佳的最终步骤， 一体化实验设计和实现这些的方法 目标弧。(1)构建HIV-1和FIV的嵌合蛋白 用于确定参与选择DNA的蛋白质结构域的整合酶 整合位点，并使用基于PCR的选择和扩增 确定整合酶最佳DNA序列的方案 认识，（2）通过研究， 体外和体内的融合活性和整合模式 由整合和序列特异性DNA结合组成的蛋白质 蛋白质，和（3）使用一种新的策略来研究病毒3 '- 和5 '-末端连接，并开发用于表征 5 ′-末端连接步骤和所需的因素。这项研究可能揭示 抑制剂的潜在新靶点，并为药物 筛选从研究整合位点选择中获得的信息 可能导致一种新的方法，在特定的位置插入外源基因， 网站.

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.