Analysis of interactions of HIV Tat and TAR with HEXIM1, 7SK, and P-TEFb

HIV Tat 和 TAR 与 HEXIM1、7SK 和 P-TEFb 的相互作用分析

• 批准号: 7337264
• 负责人: David H Price
• 金额: $ 23.63万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7337264
• 关键词: 5' Untranslated Regions; Address; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Cell Line; Cells; Complex; Condition; Coupled; Crystallization; Cyclin-Dependent Kinases; Data; Drug Formulations; Electrophoretic Mobility Shift Assay; Enhancers; Genetic Transcription; Genome; Goals; HIV; HIV Infections; HIV therapy; HIV-1; In Vitro; Investigation; Laboratories; Methods; Modeling; Molecular; Nucleic Acids; Phase; Phosphotransferases; Polymerase; Positioning Attribute; Positive Transcriptional Elongation Factor B; Price; Process; Production; Properdin; Protein Binding; Protein Overexpression; Proteins; RNA; RNA-Binding Proteins; Range; Recruitment Activity; Regulation; Reporter Genes; Response Elements; Small Nuclear RNA; Staging; Structure; Techniques; Trans-Activators; Transactivation; Transcript; Untranslated Regions; Viral; Viral Proteins; Work; base; cell type; experience; in vitro Assay; in vivo; prevent; programs; promoter; protein structure; research study; response; structural biology; success; therapy design

DESCRIPTION (provided by applicant): The AIDS virus, HIV, utilizes the host cell factor P-TEFb to regulate its transcriptional and replication program. A small viral protein, Tat, and the 5' untranslated region (UTR) of the viral transcript are both involved in this process. Tat is an RNA binding protein that binds to the trans-activation response element, TAR, that is present in the 5' UTR. Tat can form a complex with TAR and P-TEFb. P-TEFb is a cyclin dependent kinase that controls the elongation phase of transcription, by regulating the transition into productive elongation. P-TEFb itself is regulated by the reversible association of a cellular RNA binding protein HEXIM1 or HEXIM2 and the small nuclear RNA, 7SK. This large form P-TEFb is relatively inactive compared to the free form of P-TEFb, but represents the major form of P-TEFb in many cell types. Therefore, most of the potential P-TEFb activity is held in this large complex. The original model for Tat transactivation emphasized the recruitment of P-TEFb to the nascent viral transcript; however, data recently obtained suggest that HIV utilizes the cellular P-TEFb control machinery more extensively than previously imagined. We plan to examine these broader regulatory mechanisms using a combination of biochemical and structural biological techniques. In this collaborative effort between a biochemistry lab and a structural biology lab, existing in vitro assays will be used to find conditions to generate relevant complexes containing proteins, or proteins and RNA that will form crystals. Finally the structures of these complexes will be solved using crystallographic techniques. The results from these studies will ultimately yield structural and functional information needed for the rational design of therapies targeting HIV, and will provide a basis for further understanding control of cellular transcription by P-TEFb. This project is aimed at understanding how HIV utilizes cellular P-TEFb regulatory machinery to control its own transcriptional program with the prospect of developing rational therapies for HIV. Complexes comprised of viral and cellular proteins and RNAs will be crystallized and structures determined.

描述（由申请人提供）：艾滋病病毒，HIV使用宿主细胞因子P-TEFB来调节其转录和复制程序。病毒转录本的小病毒蛋白，TAT和5'未翻译区（UTR）都参与了这一过程。 TAT是一种与5'UTR中存在的反式激活响应元件TAR结合的RNA结合蛋白。 TAT可以与TAR和P-TEFB形成复合物。 P-TEFB是一种细胞周期蛋白依赖性激酶，通过调节转变为生产伸长来控制转录阶段。 P-TEFB本身受细胞RNA结合蛋白Hexim1或Hexim2和小核RNA（7SK）的可逆关联来调节。与P-TEFB的自由形式相比，这种大型P-TEFB相对不活跃，但代表了许多细胞类型中P-TEFB的主要形式。因此，大多数潜在的P-TEFB活性都在这个大型复合物中持有。 TAT反式激活的原始模型强调了P-TEFB募集到新生病毒转录本。但是，最近获得的数据表明，HIV比以前想象的更广泛地利用细胞P-TEFB控制机制。我们计划使用生化和结构生物学技术的结合来检查这些更广泛的调节机制。在生物化学实验室和结构生物学实验室之间的这种协作工作中，现有的体外测定将用于寻找条件，以生成含有蛋白质的相关复合物，或将形成晶体的蛋白质和RNA。最后，这些配合物的结构将使用晶体学技术解决。这些研究的结果最终将产生针对HIV的疗法的合理设计所需的结构和功能信息，并将为进一步理解P-TEFB对细胞转录的控制提供基础。该项目的目的是了解HIV如何利用细胞P-TEFB调节机制来控制自己的转录计划，以开发艾滋病毒的理性疗法。由病毒和细胞蛋白和RNA组成的复合物将结晶并确定结构。