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SUBSTRATE INTERACTIONS OF A MULTIFUNCTIONAL INTEGRASE

多功能整合酶的底物相互作用

基本信息

批准号：
6087471
负责人：
MICHAEL KATZMAN
金额：
$ 31.02万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-04-15 至 2002-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6087471
关键词：
DNA Lentivirus chimeric proteins enzyme activity enzyme substrate human immunodeficiency virus 1 integrase intermolecular interaction tissue /cell culture virus DNA virus integration

项目摘要

Integration of a copy of the retroviral genome into cellular DNA is necessary for viral replication, can serve as a model for other recombination events, and offers an attractive target for specific antiviral therapy. To mediate integration in vivo, the integrase protein must catalyze two endonuclease reactions. In both cases, the nucleophilic oxygen of an OH group attacks and joins to target DNA. However, the reactions exhibit dramatically different specificities: one uses diverse nucleophiles and a single target site on viral DNA, the other uses one specific nucleophile and almost any target site on host DNA. The objectives of this plan are to learn how integrase interacts with its various substrates and to establish if perturbing these interactions blocks virus replication. The central hypothesis is that the enzyme s central region has distinct sites for viral DNA, host DNA, and the attacking nucleophilic group. This hypothesis is based on the ability of the isolated central domain to catalyze nonspecific alcoholysis (a recently discovered activity that mimics the enzyme s biological actions), which proved that this domain interacts with nucleophiles and nonviral DNA. Moreover, the central region recognizes viral DNA, as revealed by studies of chimeric human immunodeficiency virus type 1/visna virus integrase proteins. The rationale for focusing on substrate recognition sites is that this information will complement structural data to model integration and will identify new antiviral targets. The Specific Aims are to: (1) Understand how integrase interacts with its nucleophilic substrates. The unique perspective of the nonspecific alcoholysis assay will reveal how the structures of the nucleophile or target and the divalent metal cofactor affect these interactions. In addition, the nucleophile site (which is hypothesized to be near the active site in the middle of the central domain) will be identified by examining these interactions using proteins with specific amino acid substitutions. (2) Understand how integrase interacts with its target DNA substrates. Assays of new chimeric integrases will localize the viral DNA site (which is hypothesized to be in the C-terminal portion of the central domain) and the host DNA site (which is hypothesized to be in the N-terminal portion of the central domain). (3) Establish whether interfering with the interactions between integrase and its various substrates is a viable antiviral strategy. Integrase mutations that do not abolish activity but affect nucleophile choice, viral DNA specificity, or target-site selection will be placed into viruses to test the hypothesis that these interactions are rate-limiting for virus replication, i.e., that partial interference will inhibit replication.

将逆转录病毒基因组的拷贝整合到细胞DNA中对于病毒复制是必需的，可以作为其他重组事件的模型，并且为特异性抗病毒治疗提供了有吸引力的靶标。为了介导体内整合，整合酶蛋白必须催化两个核酸内切酶反应。在这两种情况下，OH基团的亲核氧攻击并连接到靶DNA。然而，这些反应表现出显着不同的特异性：一种使用不同的亲核试剂和病毒DNA上的单个靶位点，另一种使用一种特定的亲核试剂和宿主DNA上的几乎任何靶位点。本计划的目标是了解整合酶如何与其各种底物相互作用，并确定干扰这些相互作用是否会阻止病毒复制。核心假设是，酶的中心区域有不同的位点，病毒DNA，宿主DNA和攻击亲核基团。这一假设是基于分离的中心结构域催化非特异性醇解的能力（最近发现的一种模拟酶生物学作用的活性），这证明了该结构域与亲核试剂和非病毒DNA相互作用。此外，中心区域识别病毒DNA，如嵌合人类免疫缺陷病毒1型/visna病毒整合酶蛋白的研究所揭示的。关注底物识别位点的基本原理是，这些信息将补充结构数据以建立模型整合，并将鉴定新的抗病毒靶点。具体目的是：（1）了解整合酶与其亲核底物的相互作用。非特异性醇解分析的独特视角将揭示亲核试剂或靶点的结构和二价金属辅因子如何影响这些相互作用。此外，将通过使用具有特定氨基酸取代的蛋白质检查这些相互作用来鉴定亲核位点（假设其靠近中心结构域中间的活性位点）。 (2)了解整合酶如何与其靶DNA底物相互作用。新嵌合整合酶的测定将定位病毒DNA位点（假设其位于中心结构域的C-末端部分）和宿主DNA位点（假设其位于中心结构域的N-末端部分）。 (3)确定干扰整合酶及其各种底物之间的相互作用是否是一种可行的抗病毒策略。不会消除活性但影响亲核选择、病毒DNA特异性或靶位点选择的整合酶突变将被置于病毒中，以测试这些相互作用限制病毒复制速率的假设，即，部分干扰会抑制复制