Structure and Function of Integrase

说明(申请人提供)：整合酶(IN)是逆转录病毒复制所必需的三种病毒编码酶之一，因此是制定艾滋病治疗策略的重要目标。尽管我们和其他人已经鉴定了一些具有抗病毒活性的HIV-1 IN抑制剂，但由于缺乏关于活性IN复合体的分子结构及其与病毒和宿主DNA底物相互作用的关键细节，进展受到严重限制。在这一竞争更新中描述的新方法旨在缩小这一差距，受益于为研究ASV IN而开发的实验系统。前两个目标涉及IN-DNA相互作用的特定特征：Aim 1将识别促进或稳定病毒DNA末端去配对的ASV和HIV-1 IN残基，这是通过使用化学探测、生物物理方法和突变进行处理所需的步骤。AIM 2将使用新的光交联法和IN-DNA复合体的分子模型来绘制IN与病毒和靶DNA之间的特定接触图。模拟和测试的迭代过程将确定IN和底物DNA之间的所有相关接触点。在AIM 3中，将用动态光散射和小角X射线散射方法分析ASV IN蛋白质和IN-DNA复合体，以确定它们在溶液中的组成和构象。化学捕集方法将被用来制备共价连接的、与生物相关的IN-DNA络合物，用于结构分析。这些分析的结果将证实这些络合物中IN结构域和底物DNA的化学计量和空间取向，并支持AIM 2的建模过程。将在结晶试验中测试均质络合物制剂，目的是获得原子分辨率的结构数据。从这些研究中得出的关于结构和机制的预测将用HIV-1 IN进行检验。该方案中的实验旨在提供对HIV-1 IN及其DNA底物之间的分子相互作用的详细了解。这些信息对于利用这种病毒蛋白作为设计抗艾滋病新疗法的目标至关重要。

DESCRIPTION (provided by applicant): Integrase (IN) is one of three viral-encoded enzymes that are essential for retroviral replication and, therefore, an important target for the development of strategies for the treatment of AIDS. Although some HIV-1 IN inhibitors with antiviral activity have been identified by us and others, progress has been seriously limited by a lack of critical details concerning the molecular structure of the active IN complex and its interactions with viral and host DNA substrates. The new approaches described in this competing renewal are designed to close this gap, benefiting from experimental systems developed to study ASV IN. The first two Aims address specific features of IN-DNA interactions: Aim 1 will identify ASV and HIV-1 IN residues that promote or stabilize viral DNA end unpairing, a step required for processing, by using chemical probing, biophysical methods, and mutagenesis. Aim 2 will map specific contacts between IN and viral and target DNA using new photo crosslinking methods together with molecular models of IN-DNA complexes. An iterative process of modeling and testing will identify all relevant contact sites between IN and substrate DNAs. In Aim 3 ASV IN proteins and IN-DNA complexes will be analyzed by dynamic light scattering and small angle X-ray scattering methods to determine their composition and conformation in solution. Chemical trapping methods will be used to prepare covalently linked, biologically-relevant IN-DNA complexes for structural analysis. The results of these analyses will confirm the stoichiometry and spatial orientation of IN domains and substrate DNAs in these complexes and support the modeling process of Aim 2. Homogeneous complex preparations will be tested in crystallization trials with the goal of acquiring structural data at atomic resolution. Predictions concerning structure and mechanism derived from these studies will be tested with HIV-1 IN. Experiments in this proposal are designed to provide a detailed understanding of the molecular interactions between HIV-1 IN and its DNA substrates. Such information is crucial for exploiting this viral protein as a target for the design of new therapies against AIDS.