Activity and inhibition of the TAT protein from HIV

描述(申请人提供)：HIV和相关的慢病毒已经进化出基本的调节机制，利用RNA结合蛋白来控制基因表达。一种名为TAT的蛋白质通过与病毒转录本5‘端的TAR RNA位点结合来增强转录延伸。虽然人们对其转录机制知道得很多，但人们只知道Tat-Tar复合体的一小部分结构。我们希望获得慢病毒TAT-TAR复合体的更完整的结构图，了解这种相互作用如何在病毒环境中进化，并利用这一知识来识别可能抑制TAT功能和病毒复制的高亲和力HIV-1 TAR结合蛋白。在之前的资助期间，我们发现Jembra病病毒(JDV)Tat的富含精氨酸的基序(ARM)是一个“变色龙”RNA结合域，可以通过两种不同的结合模式与HIV和BIV TAR结合-在BIV TAR上采用高亲和力的β-发夹构象，在HIV TAR上采用依赖于细胞蛋白Cyclin T1(CycT1)的扩展构象。我们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结合的大型组合肽库，这两种方法都将有助于识别新的高亲和力HIV TAR结合蛋白。此外，我们还表征了CycT1的结构域结构，使我们能够更详细地研究HIV TAR环是如何识别的。我们现在计划：1)利用RNA结合肽的组合文库来研究TAT-TAR相互作用的协同进化，并设计高亲和力结合HIV TAR的β-发夹肽；2)测试TAR结合肽和延伸抑制剂抑制TAT介导的激活和病毒复制的能力；3)表征CycT1-TAR的相互作用并确定CycT1或相关复合体的结构；4)制备不依赖金属的TAT-Cyclin T1-TAR复合体，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焦油识别提供更详细的结构洞察，将帮助我们了解人和牛Tat-Tar复合体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。

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.