Activity and inhibition of the TAT protein from HIV

HIV TAT 蛋白的活性和抑制

• 批准号: 6828292
• 负责人: ALAN D FRANKEL
• 金额: $ 33.8万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-30 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6828292
• 关键词: RNA binding protein; combinatorial chemistry; conformation; flavopiridol; gene induction /repression; green fluorescent proteins; human immunodeficiency virus 1; intermolecular interaction; kanamycin; nuclear magnetic resonance spectroscopy; peptide library; protein structure function; ribonucleosides; surface plasmon resonance; transcription factor; virus genetics; virus replication

DESCRIPTION (provided by applicant): HIV and related lentiviruses have evolved essential regulatory mechanisms that utilize RNA-binding proteins to control gene expression. One protein, Tat, enhances transcription elongation by binding to the TAR RNA site at the 5' end of the viral transcripts. While much is known about its transcriptional mechanism, the structure of only a small part of the Tat-TAR complex is known. We wish to obtain a more complete structural view of lentiviral Tat-TAR complexes, to understand how such interactions can evolve in a viral context, and to utilize this knowledge to identify high affinity HIV-1 TAR binders that may inhibit Tat function and viral replication. During the previous grant period, we discovered that the arginine-rich motif (ARM) of Jembrana disease virus (JDV) Tat is a "chameleon" RNA-binding domain that can bind HIV and bovine immunodeficiency virus (BIV) TARs in two different binding modes - adopting a high affinity beta-hairpin conformation on BIV TAR and an extended conformation on HIV TAR that depends on a cellular protein, cyclin T1 (CycT1). We have developed a viral replication assay to study the evolution of Tat-TAR interactions and a bacterial assay to screen large combinatorial peptide libraries for RNA binders, both of which will aid in identifying novel high affinity HIV TAR binders. In addition, we have characterized the domain structure of CycT1, allowing us to study in more detail how the HIV TAR loop is recognized. We now plan to: 1) Utilize combinatorial libraries of RNA-binding peptides to examine the co-evolution of Tat- TAR interactions and design beta-hairpin peptides that bind HIV TAR with high affinity; 2) Test the ability of TAR binding peptides and elongation inhibitors to inhibit Tat-mediated activation and viral replication; 3) Characterize CycT1-TAR interactions and determine the structures of CycT1 or relevant complexes; 4) Generate metal-independent Tat-cyclin T1-TAR complexes to circumvent a major roadblock to biophysical and structural studies. Our proposed experiments will provide much more detailed structural insights into TAR recognition, will help us understand the structural and evolutionary relationships between the human and bovine Tat-TAR complexes and how new RNA-binding specificities can evolve, and will provide new avenues for inhibitor design.

描述（由申请人提供）：HIV和相关慢病毒已经进化出利用RNA结合蛋白控制基因表达的基本调控机制。一种蛋白质达特通过与病毒转录物5'端的TAR RNA位点结合来增强转录延伸。虽然对它的转录机制了解很多，但只有一小部分Tat-TAR复合物的结构是已知的。我们希望获得慢病毒Tat-TAR复合物的更完整的结构视图，以了解这种相互作用如何在病毒背景下演变，并利用这些知识来鉴定可能抑制达特功能和病毒复制的高亲和力HIV-1 TAR结合剂。在之前的资助期间，我们发现Jembrana病病毒（JDV）达特的富含精氨酸的基序（ARM）是一个“变色龙”RNA结合结构域，可以以两种不同的结合模式结合HIV和牛免疫缺陷病毒（BIV）TAR-BIV TAR上采用高亲和力β-发夹构象，HIV TAR上采用依赖于细胞蛋白细胞周期蛋白T1（CycT 1）的扩展构象。我们已经开发了一种病毒复制试验来研究Tat-TAR相互作用的演变，并开发了一种细菌试验来筛选RNA结合剂的大型组合肽文库，这两者都将有助于鉴定新型高亲和力HIV TAR结合剂。此外，我们已经表征了CycT 1的结构域结构，使我们能够更详细地研究HIV TAR环是如何被识别的。我们现在计划：1）利用RNA结合肽的组合文库来检测达特- TAR相互作用的共进化，并设计以高亲和力结合HIV TAR的β-发夹肽; 2）测试TAR结合肽和延伸抑制剂抑制Tat介导的活化和病毒复制的能力; 3）表征CycT 1-TAR相互作用并确定CycT 1或相关复合物的结构; 4）产生不依赖于金属的Tat-细胞周期蛋白T1-TAR复合物，以绕过生物物理和结构研究的主要障碍。我们提出的实验将为TAR识别提供更详细的结构见解，将帮助我们了解人和牛Tat-TAR复合物之间的结构和进化关系，以及新的RNA结合特异性如何进化，并将为抑制剂设计提供新的途径。