Favored sites for HIV cDNA integration in the human genome

描述（由申请方提供）：HIV cDNA整合到人类基因组中的FXR位点。HIV的复制需要将病毒RNA基因组的DNA拷贝整合到宿主细胞染色体中。介导病毒整合酶（IN）蛋白作用的基本化学步骤现在已经很好地理解，因此兴趣转向了解细胞因子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批准的第一种整合酶（IN）抑制剂，可结合酶活性位点并阻断DNA链转移。然而，在雷特格韦存在下艾滋病毒的进化会引发耐药性突变体，从而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PSIP 1/LEDGF/p75（以下简称LEDGF），这对有效整合很重要，该奖项下的先前工作表明，LEDGF通过拴系机制将HIV整合靶向活性转录单位。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，现在可以获得针对该位点的高活性抑制剂。因此，本申请的重点是确定HIV IN和整合中重要的宿主因子之间的相互作用（目的1），以及整合酶抑制剂的研究（目的2），其双重目标是优化治疗和使用抑制剂了解机制。这两个目标的研究将部分由我们研究逆转录病毒整合靶向的基础设施驱动，其中包括完善的生物信息学管道和超过10亿个整合位点序列读取的档案。上一个融资周期的进展使我们能够提出几个令人兴奋的新假设，以在更新中进行调查。1)我们假设HIV PIC与细胞蛋白质进行一系列有序的相互作用，从细胞质开始，最终将PIC传递到细胞核中的有利整合位点。2)我们假设，一种抑制剂，结合到整合酶上的LEDGF相互作用的网站可以有更广泛的影响，对艾滋病毒的复制周期比以前认识到，发送我们在LEDGF的研究新的方向。3)我们假设雷特格韦通过增加细胞NHEJ机制介导的事件比例来改变整合靶向。因此，我们的具体目标是：目标1。PIC和细胞因子之间相互作用的机制研究，以确定潜在的抑制剂靶点。目标二。探索整合酶抑制剂的作用机制，包括靶向LEDGF结合位点和活性位点。!

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. !