STRUCTURE/FUNCTION STUDIES OF HIV RNASE H

说明：（改编自申请人的摘要）核糖核酸酶H（RNase H）是逆转录病毒生命周期的必需活性， 因此，它是一个重要的药物靶点。这个目标 建议是确定结构和功能之间的关系 在HIV-1逆转录酶的RNase H结构域中。研究 方法包括各种生物物理和遗传实验。的 已知HIV逆转录酶的分离的RNase H结构域折叠 进入RNase H样结构基序，然而， 不活跃。C端不稳定，缺少基本突起 PI假设在这种缺乏中发挥重要作用 的活动。为了获得HIV RNA酶的选择性抑制剂， 主动隔离域是必要的。此域的活动变体将 通过合理设计和遗传选择的结合产生。 申请人已经获得了一种这样的Mn++依赖性结构域 初步结果。这些活性物质的结构和稳定性 变体将通过圆二色性、荧光、NMR 和晶体结构研究。C的特殊作用-- 将进一步研究终端区域。初步研究显示， 来自HIV这一区域的肽是非结构化的， 活性E. coli同源物和MoLV的折叠良好。 突变对分离的结构域和肽两者的影响将 被定性。肽抑制剂将被设计成与 蛋白质的这个区域。试图描述和干涉 与结构/功能关系，平衡和动力学 分离的RNase H结构域的折叠特性也将被 研究了由于非对称的翻译后加工， HIV逆转录酶分子似乎需要其中一种RNase H结构域至少部分未折叠，这些研究也可能揭示 该机制的光。拟议研究的最终目的是 选择性抑制这种基本的逆转录病毒活性。

DESCRIPTION: (Adapted from Applicant's Abstract) Ribonuclease H (RNase H) is an essential activity for the lifecycle of retroviruses such as HIV, and therefore represents an important drug target. The goal of this proposal is to determine the relationship between structure and function in the RNase H domain of HIV-1 reverse transcriptase. The research approaches include a variety of biophysical and genetic experiments. The isolated RNase H domain of HIV reverse transcriptase is known to fold into a RNase H-like structural motif, yet the refolded enzyme is inactive. Instability in the C-terminus and a missing basic protrusion have been postulated by the PI to play an important role in this lack of activity. In order to obtain selective inhibitors of HIV RNase, an active isolated domain is necessary. Active variants of this domain will be generated by a combination of rational design and genetic selections. One such Mn++-dependent domain has already been obtained by the applicant in preliminary results. The structure and stability of these active variants will be characterized by circular dichroism, fluorescence, NMR and crystallographic structural studies. The special role of the C- terminal region will be investigated further. Preliminary studies reveal that peptides from this region of HIV are unstructured, whereas similar peptides from the active E. coli homologue and MoLV are well folded. Effects of mutations on both the isolated domain and the peptides will be characterized. Peptide inhibitors will be designed that compete with this region of the protein. In an attempt to characterize and interfere with the structure/function relationship, the equilibrium and kinetic folding properties of the isolated RNase H domain will also be investigated. Since the asymmetric post-translational processing of the HIV reverse transcriptase molecule appears to require one of the RNase H domains to be at least partially unfolded, these studies may also shed light on this mechanism.The ultimate aim of the proposed research is selective inhibition of this essential retroviral activity.