Biochemical Mechanism of HIV DNA Integration

描述（由申请人提供）：数百万人感染了艾滋病病毒HIV-1，在过去十年中，高效抗病毒治疗在减缓病毒传播和改善感染者预后方面取得了巨大进展。由于许多使用的抗病毒化合物会引起明显的副作用，并且病毒的快速进化导致大量耐药菌株，因此持续需要发现抑制HIV-1复制的新药。这种研究是通过对病毒生长的细胞和分子生物学步骤的详细了解来推动的。病毒复制周期的关键步骤是通过逆转录将病毒DNA整合到细胞染色体中。整合是由整合酶催化的，第一个整合酶抑制剂于2007年被批准用于临床。在此，我们将确定整合酶结构和功能的关键未知方面。我们的工作和其他人先前强调，整合酶与细胞因子密切相关，以完成病毒整合。关键因子晶状体上皮衍生生长因子（LEDGF）在整合过程中将HIV-1束缚在活性基因上，但LEDGF束缚的潜在机制基础尚不清楚。这将破译使用各种细胞生物学，生物化学和病毒学实验技术。即使在完全没有LEDGF的情况下，HIV-1仍然倾向于整合活性基因而不是随机基因，这导致了其他病毒相互作用蛋白帮助指导HIV-1寻找染色体位点的假设。因此，许多其他病毒结合蛋白在整合中的作用将被破译。虽然人们认为整合酶蛋白的四聚体是催化整合的多聚体，但缺乏详细的结构生物学信息限制了我们对其组织的概述。将进行生化和病毒学实验来确定活性整合酶四聚体的功能组织。由于LEDGF是引导病毒到达整合位点的天然纽带，新的DNA结合域将与LEDGF一起进行测试，以确定HIV-1可以被引导到新的整合位点的程度。这些实验的成功完成将揭示HIV- 1整合酶结构和功能的基本方面，这将大大有助于发现新的整合酶抑制剂。此外，它们还可能为开发慢病毒载体开辟新的途径，用于未来的基因治疗患者。

DESCRIPTION (provided by applicant): Millions of people are infected with the AIDS virus HIV-1, and highly active antiviral therapy has made great strides over the past decade to slow virus spread and improve infected individuals' prognoses. Because many of the utilized anti-viral compounds elicit significant side effects, and rapid virus evolution leads to significant numbers of drug resistant strains, there is an ongoing need to discover new drugs to inhibit HIV-1 replication. Such research is driven through detailed understanding of the cellular and molecular biological steps that the virus undergoes to grow. The pivotal step in the viral replication cycle is the integration of the viral DNA made by reverse transcription into a cell chromosome. Integration is catalyzed by integrase, and the first integrase inhibitor was approved for clinical use in 2007. Herein, we will ascertain critical unknown aspects of integrase structure and function. Our work and others have previously highlighted that integrase works in close association with cellular factors to accomplish virus integration. The key factor, lens epithelium-derived growth factor (LEDGF), tethers HIV-1 to active genes during integration, but the underlying mechanistic basis of LEDGF tethering is unknown. This will be deciphered using a variety of cell biology, biochemical, and virological experimental techniques. Even in the complete absence of LEDGF, HIV-1 still favors active genes for integration over random, leading to the hypothesis that other virus-interacting proteins help to guide HIV-1 as it seeks chromosomal sites. The roles of numerous other viral binding proteins in integration will therefore be deciphered. Though it is believed that a tetramer of the integrase protein is the multimer that catalyzes integration, a dearth of detailed structural biology information has limited our overview of its organization. Biochemical and virological experiments will be conducted to define the functional organization of the active integrase tetramer. As LEDGF is the natural tether that guides the virus to sites of integration, novel DNA binding domains will be tested in concert with LEDGF to ascertain the extent that HIV-1 can be directed to new sites for integration. The successful completion of these experiments will uncover fundamental aspects of HIV- 1 integrase structure and function, which will significantly help in the discovery of novel integrase inhibitors. They moreover may open up new ways in which to develop lentiviral vectors for future treatment of patients with genetic therapy. PUBLIC HEALTH RELEVANCE: HIV-1 replication critically relies on integrase activity, the viral enzyme that integrates the reverse transcript into a cell chromosome. This application will uncover novel aspects of integrase structure and function, which will significantly impact ongoing discovery efforts to target and block the pivotal integration step in the virus replication cycle.