STRUCTURE/FUNCTION OF INTEGRASE

逆转录病毒酶，整合酶（IN），催化插入新的 将病毒DNA合成到宿主细胞染色体中。 此事件 正常的病毒复制所必需的 因此，像HIV-1逆转 转录酶和蛋白酶，IN是一个潜在的目标，抑制在 预防或治疗艾滋病。 研究的主要目标描述 在这个建议中，是为了了解HIV-1 IN的分子结构， 单独或与其DNA底物复合，因此， 抑制剂可以建立在合理的基础上。 三个具体目标是 建议：第一个具体目标描述了不同的结构分析 结构域和全长IN，在金属辅因子的存在下，选择 模型底物抑制剂和单克隆抗体片段，使用X- 射线晶体学和核磁共振。 第二个具体目标将测试具体 结构数据所建议的智能网组织和功能模型， 使用IN的定点诱变和化学修饰。 的 第三个具体目标描述了一种新的突触末端病毒DNA的使用 底物，以研究IN如何与DNA在功能性 催化协调处理反应的复合物。 特定 预测DNA弯曲和部分解旋的模型将被测试， 使用DNA足迹技术。 本文中描述的实验 建议采用广泛最先进的分子遗传学， 生物化学和生物物理方法，以及新的底物， 免疫学试剂 他们还建立在广泛的经验， 主要研究者和人类（HIV）和禽类（ASV）合作者 病毒整合系统，以及利用ASV IN作为有价值的 比较的基础。 所获得的知识应该有助于描述 艾滋病毒特有的特征。 这些研究结果提供了 抑制HIV整合和发展的新分子靶点 艾滋病治疗。

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.