LEDGF/p7 and HIV Integration

描述（由申请人提供）：虽然整合反应的基本核酸化学被合理地理解，但病毒整合前复合物的整合在细胞核的复杂环境中实际上如何发生在许多方面仍然是神秘的。特别是，整合领域是强烈感兴趣的细胞分子如何积极或消极地参与前整合复合物和染色质之间的反应，以及它们如何决定反应发生在宿主细胞基因组中的位置和时间。转录辅激活因子LEDGF/p75现在已经被安全地暗示为慢病毒特异性的整合辅因子。一个基本的分子拴系模型已经提出，其中LEDGF/p75拴系IN染色质，关键参与蛋白质结构域已被确定，和精确的结构信息存在的LEDGF/p75结构域与IN相互作用。LEDGF/p75的假设作用包括作为催化步骤的辅因子，保护IN和其所在的整合前复合物免于降解，以及引导或束缚整合前复合物至染色质。后一种机制对于病毒参与染色质和整合本身可能是至关重要的，并且它也可能决定HIV整合位点现在明显的独特基因组分布。因此，了解LEDGF/p75对HIV生物学的重要性可能对了解潜伏期具有长期意义，并可能提供新的治疗靶点。它甚至可以为靶向慢病毒载体铺平道路，以减少插入突变的风险。在这个项目中，我们将使用有效的RNAi敲除方法、基因敲除细胞、病毒学分析、结构域定位和异源拴系技术、全基因组整合位点分析以及细胞生物学和蛋白质组学方法来进一步理解和利用LEDGF/p75的病毒生物学。本项目将利用病毒学、生物化学、基因组学和嵌合蛋白方法阐明转录辅因子LEDGF/p75在HIV-1生命周期中的重要性。

DESCRIPTION (provided by applicant): While the essential nucleic acid chemistry of the integration reaction is reasonably understood, how integration of the viral pre-integration complex actually happens in the complex environment of the cell nucleus remains mysterious in many respects. In particular, the integration field is intensely interested in how cellular molecules participate either positively or negatively in the reaction between the pre-integration complex and chromatin, and also in how they determine where and when the reaction occurs in the host cell genome. The transcriptional coactivator LEDGF/p75 has now been securely implicated as an integration cofactor specific for lentiviruses. A basic molecular tethering model has been put forth in which LEDGF/p75 tethers IN to chromatin, key participating protein domains have been identified, and precise structural information exists for the LEDGF/p75 domain that interacts with IN. Hypothesized roles of LEDGF/p75 include acting as a cofactor for catalysis steps, protecting IN and the pre- integration complex in which it resides from degradation, and guiding or tethering the pre-integration complex to chromatin. The latter mechanism could be crucial for the virus to engage chromatin and integrate per se, and it also may determine the distinctive genomic distributions now evident for HIV integration sites. Understanding the importance of LEDGF/p75 to HIV biology may therefore have long-term implications for understanding latency and may provide a new therapeutic target. It could even pave the way for targeting of lentiviral vectors to reduce insertional mutagenesis risks. In this project, we will use effective RNAi knockdown methods, genetic knockout cells, virological analyses, domain mapping and heterologous tethering techniques, genome- wide integration site analyses, and cell biological and proteomic methods to both further understand and exploit the viral biology of LEDGF/p75. PUBLIC HEALTH RELEVANCE This project will elucidate the significance of the transcriptional cofactor LEDGF/p75 in the HIV-1 life cycle, using virological, biochemical, genomic and chimeric protein approaches.