SITE-SPECIFIC TARGETING OF REGULATORY REGIONS OF HIV RNA

HIV RNA 调控区域的位点特异性靶向

• 批准号: 3096325
• 负责人: DAVID S SIGMAN
• 金额: $ 59.22万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3096325
• 关键词: AIDS; human immunodeficiency virus 1; virus RNA

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.

六名调查员在该计划项目中寻求支持以探索该地点 - 药物、肽和蛋白质与关键的特定相互作用 使用结构技术研究 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 用于抗病毒药物的设计。 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。 这些的结构模型 相互作用也将基于使用化学的足迹研究 核酸酶和肽调节区的靶向切割 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。 如果有效抑制，它们作为抗病毒药物的潜力将被探索 被观察到。 c) 跨显性大鼠突变体的特征在于它们的相互作用 具有 TAR 和 TAR 结合细胞蛋白 TRP-185 动态 3D 轮廓 TRP-185分析和TAT及其计算结构研究 将进行衍生品。 d) 针对寄生虫和病毒病原学的基于 PCR 的单一检测 将设计血液中的试剂来监测外来疾病的进展 HIV患者的感染。 它将基于之前的测定 为克氏锥虫开发，依赖于核酸酶活性 1,10-菲咯啉-铜将 DNA 切割成扩增片段。 格兰特-=P01GM395580006 该提案解决了突变 tat 蛋白抑制的机制 野生型 tat 蛋白激活 HIV-1 基因表达。 我们的 实验室证明所谓的显性失活或反显性 tat突变体可以通过引入取代或截短来构建 进入 tat 的基本域。 其中一些突变体能够抑制 当两种蛋白质以等摩尔存在时，野生型 tat 起作用 浓度。 因此，此类突变体可能具有 治疗 HIV-1 感染的治疗潜力。 然而它会 对于确定这种抑制的机制至关重要 了解 tat 功能并开发更好的 tat 功能抑制剂。 该提案的具体目标是：（1）创建额外的 转显性 tat 突变体 (2) 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体 tat 痘苗病毒和细菌表达系统中的蛋白质以测试其 改变 HIV-1 基因表达的能力 (4) 确定 与野生型和反式显性 tat 相关的细胞蛋白 蛋白质。 这些研究应该有助于更好地理解 并为tat抑制剂的开发提供基础 功能。