SITE-SPECIFIC TARGETING OF REGULATORY REGIONS OF HIV RNA

六名研究人员寻求本计划项目的支持，以探索该网站- 药物、肽和蛋白质与关键的 HIV RNA的调控区域。 在分子水平上理解这些过程将提出新的目标 设计抗病毒药的方法 a）将通过以下方法研究tat蛋白-TAR和rev蛋白-RRE相互作用： X射线晶体学和高分辨率NMR。 这些结构模型 相互作用也将基于使用化学物质的足迹研究。 核酸酶和肽对调节区的靶向切割 与1，10-菲咯啉-铜连接。 B）转录和逆转录酶的序列特异性抑制剂 将根据动力学、化学和晶体学结果设计。 它们作为抗病毒剂的潜力将被探索，如果有效抑制 被观察到。 c）反式显性大鼠突变体的特征在于它们之间的相互作用 使用TAR和TAR结合细胞蛋白TRP-185动态3D剖面 TRP-185的分析和TAT及其衍生物的计算结构研究 衍生品将继续。 d）用于寄生虫和病毒病因学疾病的单一基于PCR的测定 将设计血液中的试剂来监测外源性 艾滋病患者的感染。 它将基于先前的分析， 克氏锥虫的核酸酶活性， 1，10-菲咯啉-铜将DNA切割成扩增片段。 授予-= P01GM395580006 该提案提出了突变tat蛋白抑制 野生型tat蛋白激活HIV-1基因表达。 我们 实验室证明，所谓的显性阴性或转显性 可以通过引入取代或截短 tat的基本领域。 其中一些突变体能够抑制 当两种蛋白质以等摩尔浓度存在时， 浓度的 因此，这些突变体可能具有 在治疗HIV-1感染方面的治疗潜力。 然而将 关键是要确定这种抑制的机制， 了解tat功能并开发更好的tat功能抑制剂。 这项建议的具体目标是：（1）增加 反式显性tat突变体（2）来构建重组HIV-1病毒， 含有反式显性tat突变体（3）以产生野生型和突变tat 在牛痘病毒和细菌表达系统中的蛋白质，以测试它们的 改变HIV-1基因表达的能力（4），以确定 与野生型和反式显性tat相关的细胞蛋白 proteins. 这些研究应该会使我们更好地了解tat 为开发Tat抑制剂提供了基础 功能

Six investigators seek support in this Program Project to explore the site- specific interactions of drugs, peptides and proteins with the key regulatory regions of HIV RNA using structural techniques. Understanding these processes on a molecular level will suggest new targets for the design of antiviral agents. a) The tat protein-TAR and rev protein-RRE interaction will be studied by X-ray crystallography and high resolution NMR. Structural models for these interactions will also be based on footprinting studies using chemical nucleases and the targeted scission of the regulatory regions by peptide linked to 1,10-phenanthroline-copper. b) Sequence-specific inhibitors of transcription and reverse transcriptase will be devised based on kinetic, chemical and crystallographic results. Their potential as antiviral agents will be explored if potent inhibition is observed. c) Transdominant rat mutants will be characterized by their interaction with TAR and the TAR binding cellular protein TRP-185 Dynamic 3D Profile Analysis of TRP-185 and computational structural studies of TAT and its derivatives will be carried. d) A single PCR-based assay for parasitic and viral etiological disease agents in blood will be devised to monitor the progression of adventitious infection in HIV patients. It will be based on a assay previously developed for Trypanosoma cruzi which relies on the nuclease activity of 1,10-phenanthroline-copper to cut DNA into amplified fragments. GRANT-=P01GM395580006 This proposal addresses the mechanisms by which mutant tat proteins inhibit wild-type tat proteins from activating HIV-1 gene expression. Our laboratory demonstrated that so called dominant negative or transdominant tat mutants can be constructed by introducing substitutions or truncations into the basic domain of tat. Several of these mutants are able to inhibit wild-type tat function when both proteins are present in equimolar concentrations. Thus, it is possible that such mutants may have therapeutic potential in the treatment of HIV-1 infection. However it will be critical to determine the mechanism of this inhibition both the understand tat function and to develop better inhibitors of tat function. This proposal has as its specific aims: (1) to create additional transdominant tat mutants (2) to construct recombinant HIV-1 viruses that contain transdominant tat mutants (3) to produce wild-type and mutant tat proteins in vaccinia virus and bacterial expression systems to test their ability to alter HIV-1 gene expression (4) to determine the ability of cellular proteins to associate with wild-type and transdominant tat proteins. These studies should lead to a better understanding of tat function and provide the basis for the development of inhibitors of tat function.