Favored sites for HIV cDNA integration in the human genome

描述(申请人提供)：人类基因组中HIV基因整合的有利位置。摘要复制HIV需要将病毒RNA基因组的DNA拷贝整合到宿主细胞染色体中。病毒整合酶(IN)蛋白调节作用的基本化学步骤现在已被很好地了解，因此人们的兴趣转向了解细胞因素在体内整合过程中的作用，并利用这些信息开发抑制剂。Raltegravir是FDA批准的第一个整合酶(IN)抑制剂，它结合酶活性部位并阻止DNA链转移。在Raltegravir存在的情况下，HIV的进化引起了耐药突变，然而，这激发了对整合前复合体中潜在新药靶点的研究。IN结合宿主细胞蛋白PSIP1/LEDGF/p75(以下简称LEDGF)，这对有效整合非常重要，该奖项下的先前工作表明，LEDGF通过一种拴系机制将HIV整合到活跃的转录单位。我们还报道了IN上的LEDGF结合位点可以被小分子抑制剂结合，现在已经有了针对该位点的高活性抑制剂。因此，这项应用侧重于确定HIV IN与整合中重要的宿主因素之间的相互作用(目标1)，以及整合酶抑制剂的研究(目标2)，具有优化治疗和使用抑制剂了解机制的双重目标。这两个目标的研究将在一定程度上受到我们研究逆转录病毒整合靶向的基础设施的推动，该基础设施包括完善的生物信息管道和超过10亿个整合位点序列读取的档案。上一个资金周期的进展使我们能够提出几个令人振奋的新假设，以在更新中进行研究。1)我们假设，HIV PIC从细胞质开始，与细胞蛋白进行一系列有序的相互作用，最终将PIC传递到细胞核中受欢迎的整合位置。2)我们假设，与整合酶上的LEDGF相互作用位点结合的抑制剂对HIV复制周期的影响可能比先前认识的更广泛，这为我们在LEDGF的研究中提供了新的方向。3)我们假设raltegravir通过增加由细胞NHEJ机制介导的事件的比例来改变整合靶向。因此，我们的具体目标是：目标1。从机制上研究PIC和细胞因子之间的相互作用，以确定潜在的抑制靶点。目的2.探讨针对LEDGF结合部位和活性部位的整合酶抑制剂的作用机制。好了！

DESCRIPTION (provided by applicant): Favored sites for HIV cDNA integration in the human genome. Abstract Replication of HIV requires integration of a DNA copy of the viral RNA genome into a host cell chromosome. The basic chemical steps mediating action of the viral integrase (IN) protein are now well understood, so interest turns to understanding the roles of cellular factors in the integration process in vivo, and use of such information to develop inhibitors. Raltegravir, the first FDA-approved integrase (IN) inhibitor, binds the enzyme active site and blocks DNA strand transfer. Evolution of HIV in the presence of raltegravir elicits resistance mutants, however, motivating studies of potential new drug targets in the preintegration complex. IN binds the host cell protein PSIP1/LEDGF/p75 (henceforth LEDGF), which is important for efficient integration, and previous work under this award showed that LEDGF targets HIV integration to active transcription units via a tethering mechanism. We also reported that the LEDGF binding site on IN can be bound by small molecule inhibitors, and now highly active inhibitors targeting this site are available. This application thus focuses on identifying interactions between HIV IN and host factors important in integration (Aim 1), and studies of integrase inhibitors (Aim 2) with the dual goals of optimizing therapy and using inhibitors to understand mechanism. Research in both aims will be driven in part by our infrastructure for studying retroviral integration targeting, which consists of well established bioinformatic pipelines and an archive of over one billion integration site sequence reads. Progress from the previous funding cycle allows us to propose several exciting new hypotheses to investigate in the renewal. 1) We hypothesize that HIV PICs engage in an orderly series of interactions with cellular proteins, beginning in the cytoplasm, that ultimately delivers PICs to favored integration sites in the nucleus. 2) We hypothesize that an inhibitor that binds to the LEDGF interacting site on integrase can have wider effects on the HIV replication cycle than previously appreciated, sending us in new directions in the study of LEDGF. 3) We hypothesize that raltegravir alters integration targeting by increasing the proportion of events mediated by the cellular NHEJ machinery. Thus our Specific Aims are: Aim 1. Mechanistic studies of interactions between PICs and cellular factors to identify potential inhibitor targets. Aim 2. Probing the mechanism of integrase inhibitors, both those targeting the LEDGF binding site and the active site. !