STRUCTURE/FUNCTION OF INTEGRASE

逆转录病毒酶整合酶(IN)催化新的插入 将病毒DNA合成到宿主细胞染色体中。这项活动是 是正常病毒复制所必需的。因此，像HIV-1逆转一样 转录酶和蛋白水解酶，IN是潜在的抑制靶点 艾滋病的预防或治疗。所述研究的主要目标 在这项建议中是为了了解HIV-1 IN的分子结构， 单独和与其DNA底物形成复合体，因此设计 抑制剂可能建立在理性的基础上。三个具体目标是 建议：第一个具体目标是描述不同结构的分析 结构域和全长In，在金属辅因子存在的情况下，选择 模型底物抑制剂和单抗片段，使用X- 射线结晶学和核磁共振。第二个特定目标将测试特定的 结构数据建议的智能网组织和功能模型， 使用IN的定点突变和化学修饰。这个 第三个特定目标描述了一种新的、突触末端病毒DNA的使用 底物研究IN与DNA在功能上的相互作用 催化协调处理反应的复合体。一种特定的 预测DNA弯曲和部分解离的模型将得到测试， 使用DNA足迹技术。本文件中描述的实验 该提案使用了广泛的最先进的分子遗传学， 生化和生物物理方法，以及新的底物和 免疫试剂。他们还建立在丰富的经验基础上 PI及其与人(HIV)和禽类(ASV)的合作者 病毒集成系统，以及利用ASV IN作为有价值的 比较的基数。所获得的知识应该有助于描述 HIV特有的特征。这些研究的结果提供了 抑制HIV整合和发育的新分子靶点 艾滋病的治疗方法。

The retroviral enzyme, integrase (IN), catalyzes the insertion of newly synthesized viral DNA into the host cell chromosome. This event is required for normal viral replication. Thus, like HIV-1 reverse transcriptase and protease, IN is a potential target for inhibition in prevention or treatment of AIDS. The major goal of research described in this proposal is to understand the molecular structure of HIV-1 IN, alone and in complex with its DNA substrates, so that design of inhibitors may build upon a rational foundation. Three Specific Aims are proposed: The first Specific Aim describes structural analyses of distinct domains and full length IN, in the presence of metal cofactors, selected model substrate inhibitors and monoclonal antibody fragments, using X- ray crystallography and NMR. The second Specific Aim will test specific models of IN organization and function suggested by the structural data, using site-directed mutagenesis and chemical modification of IN. The third Specific Aim describes the use of a novel, synapsed-end viral DNA substrate to investigate how IN interacts with DNA in a functional complex that catalyzes coordinated processing reactions. A specific model that predicts DNA bending and partial unwinding will be tested, using DNA footprinting techniques. Experiments described in this proposal employ a broad range of state-of-the-art molecular genetic, biochemical, and biophysical methods, as well as novel substrates and immunological reagents. They also build upon the extensive experience of the PI and collaborators with both the human (HIV) and avian (ASV) viral integration systems, and the ability to utilize ASV IN as a valuable base of comparison. The knowledge gained should help to delineate features that are specific to HIV. The results of these studies provide new molecular targets for inhibition of HIV integration and development of AIDS therapies.