Molecular Mechanisms of HIV-1 DNA Integration

描述(申请人提供)：人类免疫缺陷病毒1型(HIV-1)和其他逆转录病毒生命周期中的一个基本步骤是将病毒基因组的双链DNA拷贝整合到宿主细胞染色体中。逆转录病毒DNA的整合是由病毒蛋白整合酶催化的。这个过程是非特定的，但也不是随机的，一些整合站点使用的频率比其他站点高得多。整合位点对入侵病毒和易感宿主都有重要影响，而决定靶点选择的机制还不是很清楚。这项提议的长期目标是进一步了解HIV-1整合的机制和控制整合宿主DNA位点选择的过程，并设计出一种指导整合到预定位点的方法。其具体目的是(1)检测HIV-1整合位点在人类基因组中的分布和偏好，并确定影响靶点选择的病毒因素和宿主条件；(2)利用整合酶融合到序列特异性DNA结合蛋白上，研究逆转录病毒DNA的定点整合。在目标1中，将建立HIV-1整合位点的文库，以检测可能影响目标位点选择的病毒或宿主因素，并确定这种改变是否会导致重大的生物学结果。HIV-1整合到某些染色体位置会优先导致潜伏期的假设将得到检验，潜伏期是目前治疗方法无法根除病毒的主要因素。为了加快分析速度，将开发一种新的高通量分析方法来确定整合位点。目的2将分析一类新型整合酶融合蛋白在培养细胞中介导定点整合的效率和特异性。这些融合蛋白由HIV-1整合酶和多指锌指E2C组成，多指锌指E2C是一种人工合成的DNA结合蛋白，以高亲和力与18bp的序列特异性结合。这些合成的蛋白质也可以被修饰，以潜在地识别任何所需的序列。还将测试一种新的策略，即通过蛋白质-蛋白质相互作用将HIV-1整合酶-供体DNA复合体输送到感兴趣的DNA位置。高效和高通量检测整合位点的分布和偏好将有助于更好地了解逆转录病毒感染过程中靶点的选择，并对插入突变的相对风险提供更好的评估。利用融合蛋白进行定点整合的研究可能带来一种在特定位置插入外源基因的新方法，这将作为一种实验工具得到广泛应用，并改善现有逆转录病毒载体的基因输送治疗应用。

DESCRIPTION (provided by applicant): An essential step during the life cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses is integration of a double-stranded DNA copy of the viral genome into a host cell chromosome. Integration of retroviral DNA is catalyzed by the viral protein integrase. The process is non-specific but is not random, with some integration sites used at much greater frequencies than others. The site of integration has important consequences to both the invading virus and the susceptible host, and the mechanism that determines target site selection is not well understood. The long-term objective of this proposal is to further understand the mechanism of HIV-1 integration and the process that controls the selection of host DNA sites for integration, and to devise a method of directing integration into a predetermined site. The specific aims are (1) to examine the distribution and preference of HIV-1 integration sites in the human genome and identify viral factors and host conditions that affect target site selection, and (2) to study site-directed integration of retroviral DNA using integrase fused to a sequence-specific DNA-binding protein. In Aim 1, libraries of HIV-1 integration sites will be generated to detect viral or host factors that can influence target site selection and determine whether the alteration can cause significant biological outcomes. The hypothesis that HIV-1 integration into certain chromosomal sites can preferentially lead to latency, a major contributing factor to the inability of current treatments to eradicate the virus, will be tested. To expedite the analysis, a new high-throughput assay for determining integration sites will be developed. Aim 2 will analyze the efficiency and specificity of a new class of integrase fusion proteins to mediate site-directed integration in cultured cells. These fusion proteins consist ofHIV-1 integrase and a polydactyl zinc-finger E2C, a synthetic DNA-binding protein that specifically binds to an 18-bp sequence with high affinity. These synthetic proteins can also be modified to potentially recognize any desired sequences. A new strategy in which the HIV-1 integrase-donor DNA complex is delivered to a DNA site of interest through protein-protein interactions will also be tested. The availability of an efficient and high throughput assay for examining the distribution and preference of integration sites will yield a better understanding of target site selection during retroviral infection and provide a better assessment on the relative risk of insertional mutagenesis. Studies on site-directed integration using fusion proteins may lead to a new approach for inserting exogenous genes at specific sites, which will have a wide application as an experimental tool and improve the therapeutic application of current retrovirus-based vectors for gene delivery.