SUBSTRATE INTERACTIONS OF A MULTIFUNCTIONAL INTEGRASE

将逆转录病毒基因组拷贝整合到细胞DNA中是病毒复制所必需的，可以作为其他重组事件的模型，并为特定的抗病毒治疗提供了一个有吸引力的靶点。为了介导体内整合，整合酶蛋白必须催化两个内切酶反应。在这两种情况下，OH基团的亲核氧攻击并连接到目标DNA上。然而，这两种反应表现出截然不同的特异性：一种反应使用多种亲核试剂和病毒DNA上的单一靶点，另一种反应使用一种特定的亲核试剂和宿主DNA上的几乎任何靶点。该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。核心假设是该酶的中心区域有不同的病毒DNA、宿主DNA和攻击的亲核基团的位置。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（一种最近发现的模仿酶的生物作用的活性），这证明了该结构域与亲核试剂和非病毒DNA相互作用。此外，正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那样，中央区域识别病毒DNA。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来建模整合，并将确定新的抗病毒靶点。具体目标是：(1)了解整合酶如何与其亲核底物相互作用。非特异性醇解分析的独特视角将揭示亲核试剂或目标和二价金属辅因子的结构如何影响这些相互作用。此外，亲核位点（假设在中心结构域中间的活性位点附近）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确定。(2)了解整合酶如何与其靶DNA底物相互作用。新的嵌合整合酶的检测将定位病毒DNA位点（假设在中心结构域的c端部分）和宿主DNA位点（假设在中心结构域的n端部分）。(3)确定干扰整合酶与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。整合酶突变不会破坏活性，但会影响亲核试剂的选择、病毒DNA特异性或靶标位点的选择，这些突变将被放入病毒中，以检验这些相互作用限制病毒复制的假设，即部分干扰将抑制复制。

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.