Protein-Protein Interactions of HIV-1 Integrase

超出提供的空间。该项目的长期目标是阐明宿主-病毒的动态相互作用，特别是整合酶(IN)和INI1/hSNF5，并利用这些知识设计抗艾滋病治疗的药物。INI1是染色质重塑SWI/SNF复合体的核心成分，也是肿瘤抑制因子。INI1/hSNF5被特异性地整合到HIV-1病毒粒子中，INI1/hSNF5显性截断突变体负向抑制HIV-1的复制。INI1/hSNF5含有一个隐蔽的核输出信号，该信号介导依赖hCRM1/exportin1的核输出。我们发现INI1/hSNF5还存在于另一个新的复合体中，与SWI/SNF复合体不同，SWI/SNF复合体的成分被整合到HIV-1病毒粒子中。我们假设，在组装和颗粒生产过程中，INI1/hSNF5及其新型核复合体被信号传递到细胞质，对接到Gag-Pol的IN部分，并组装成HIV-1病毒粒子。在靶细胞中，INI1复合体可能促进前病毒的整合和/或调节转录。在这项建议中，在特定的目的I中，使用RNA干扰和反式显性突变，我们将确定INI 1和SAP 18是否是HIV-1在生产者细胞或靶细胞中复制的早期和晚期事件所必需的。在特定的目标II中，我们将对INIL结合蛋白(如SAP18)及其与HIV-1病毒粒子的整合机制进行研究。我们将纯化新的INI1-SAP18多蛋白复合体，并测定其对IN的依赖性。在特定目的III中，我们将进行生化和遗传分析，以确定INI1及其相关蛋白如SAP18对HIV-1复制的功能意义。我们将测试纯化的INI-1及其复合体对体外整合的影响，分离和鉴定IN、INI-1和SAP-18的相互作用缺陷突变体及其分子内和分子间、等位基因特异性和非等位基因特异性回复株。在特定的目标IV中，我们将确定INI1/hSNF5的核输出特性是否对于掺入HIV-1病毒粒子以及跨显性抑制早期和晚期事件是否重要。我们的建议将全面阐述INIL相关多蛋白复合体与HIV-1募集的机制和意义，并阐明IN与其宿主蛋白的高级结构/功能关系。了解宿主-病毒相互作用的生命力可能为设计有效的抗艾滋病毒-1药物提供新的目标和见解。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. The long-term objective of this project is aimed at elucidating the dynamic host-virus interactions, specifically focusing on integrase (IN) and INI1/hSNF5, and to utilize this knowledge for the design of drugs for anti-AIDS therapy. INI1 is a core component of the chromatin remodeling SWI/SNF complex and a tumor suppressor. INI1/hSNF5 is specifically incorporated into the HIV-1 virions and a truncation mutant of INI1/hSNF5 dominant negatively inhibits HIV-1 replication. INI1/hSNF5 harbors a masked nuclear export signal that mediates the hCRM1/exportinl dependent nuclear export. We have found that INI1/hSNF5 is also present in another novel complex, distinct from the SWI/SNF complex, the component of which is incorporated into HIV-1 virions. We hypothesize that during assembly and particle production, INI1/hSNF5, along with its novel nuclear complex, is signaled to get exported to the cytoplasm, docks itself onto IN portion of Gag-Pol, and gets assembled into the HIV-1 virions. In the target cells, INI1 complex may facilitate the integration and/or regulate transcription of the provirus. In this proposal, in the specific aim I, using RNA interference and trasdominant mutants we will determine if INI 1 and SAP 18 are required for early and late events of HIV-1 replication in producer cells or target cells. In the specific aim II, we will characterize INIl-assocated proteins (such as SAP18) and mechanism of their incorporation into HIV-1 virions. We will purify the novel INI1-SAP18 multi-protein complex and determine the dependence on IN. In the specific aim III, we will carry out biochemical and genetic analysis to determine the functional significance of INI1 and its associated proteins such as SAP18 to HIV-1 replication. We will test the effect of purified INI 1 and complexes on in vitro integration, isolate and characterize interaction defective mutants of IN, INI 1 and SAP 18 and their intra-molecular and inter-molecular, allele specific and non-allele specific revertants. In the specific aim IV, we will determine if nuclear export property of INI1/hSNF5 is important for incorporation into HIV-1 virions and for transdominant inhibition of early and late events. Our proposal will provide a comprehensive account of mechanism and significance of recruitment of INIl-associated multi-protein complex to HIV-1 and elucidate the higher order structure/function relationship of IN with its host proteins. Understanding the vitality of host-virus interactions is likely to provide novel targets and insights for designing effective antivirals against HIV-1. PERFORMANCE SITE ========================================Section End===========================================