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HIV-1 Integration

HIV-1整合

基本信息

批准号：
6892130
负责人：
DUANE P GRANDGENETT
金额：
$ 25.73万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-09-01 至 2008-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6892130
关键词：
Escherichia coli enzyme mechanism human immunodeficiency virus 1 integrase molecular cloning murine leukemia virus nucleic acid repetitive sequence protein reconstitution recombinant proteins virus DNA virus assembly virus infection mechanism

项目摘要

Significant progress towards understanding the mechanisms involved in retrovirus integration is evident, but many facets of retrovirus integration remain unclear. The multimeric state of integrase (IN) subunits, their role in assembly and their molecular interactions with viral DNA in preintegration complexes (PIC) found in virus-infected cells are still undefined. The spatial arrangements of IN subunits near the viral DNA termini necessary to mediate assembly and concerted integration of the viral DNA termini are unknown. The regions of IN responsible for contact between the two viral termini bound by IN mediating concerted integration are unknown. Our initial efforts with non-ionic detergent disrupted HIV-1 virions to characterize concerted integration in vitro; has led to our recent success with newly-folded recombinant HIV-1 IN. Co-expression of HIV-1 IN with a plasmid expressing protein chaperones (GroEL-GroES) in bacteria allows purified recombinant HIV-1 IN to efficiently mediate concerted (full-site) integration without auxiliary viral or cellular proteins. We will use the reconstitution assay to determine what role newly-folded HIV-1 IN plays in specific assembly steps, in IN-LTR DNA interactions at the molecular level, in full-site integration properties of nucleoprotein complexes having characteristics of purified HIV-1 and murine leukemia virus PIC (or intasomes). The interactions of HIV-1 IN with wild type and mutant viral termini for full-site integration will be investigated. We will attempt to establish the multimeric state of HIV-1 IN subunits necessary and sufficient in nucleoprotein complexes mediating full-site integration. We will determine what amino acid residues of HIV-1 IN contribute to the protein-protein and protein-DNA interactions responsible for assembly and full-site integration. We will use the reconstituted HIV-1 IN-LTR donor complexes to investigate the effects of active site inhibitors to HIV-1 IN integration in vivo and in vitro. We will continue to investigate the role HIV-1 IN has in the retrovirus life cycle with emphasis on understanding full-site integration.

在理解逆转录病毒整合的机制方面取得了重大进展，但逆转录病毒整合的许多方面仍不清楚。整合酶亚基的多聚体状态、它们在装配中的作用以及它们在病毒感染细胞中发现的整合前复合物（PIC）中与病毒DNA的分子相互作用仍然不确定。病毒DNA末端附近的IN亚基的空间排列是介导病毒DNA末端组装和协同整合所必需的，目前尚不清楚。负责IN介导协同整合的两个病毒末端之间接触的IN区域尚不清楚。我们最初的努力与非离子去污剂破坏HIV-1病毒粒子的特点一致的整合在体外，导致我们最近的成功与新折叠的重组HIV-1 IN。HIV-1 IN与表达蛋白伴侣的质粒（GroEL-GroES）在细菌中的共表达允许纯化的重组HIV-1 IN在没有辅助病毒或细胞蛋白的情况下有效地介导协同（全位点）整合。我们将使用重建试验来确定新折叠的HIV-1 IN在特定的组装步骤中、在分子水平上的IN-LTR DNA相互作用中、在具有纯化的HIV-1和鼠白血病病毒PIC（或整合体）特征的核蛋白复合物的全位点整合特性中起什么作用。将研究HIV-1 IN与野生型和突变型病毒末端的相互作用，以进行全位点整合。我们将尝试建立HIV-1 IN亚基的多聚体状态，这是核蛋白复合物介导全位点整合所必需的。我们将确定HIV-1 IN的哪些氨基酸残基有助于蛋白质-蛋白质和蛋白质-DNA相互作用，负责组装和全位点整合。我们将使用重组的HIV-1 IN-LTR供体复合物来研究活性位点抑制剂在体内和体外对HIV-1 IN整合的影响。我们将继续研究HIV-1 IN在逆转录病毒生命周期中的作用，重点是了解全位点整合。