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

描述（由申请人提供）：艾滋病病毒HIV利用宿主细胞因子P-TEFb来调节其转录和复制程序。小病毒蛋白达特和病毒转录物的5'非翻译区（UTR）均参与该过程。达特是一种RNA结合蛋白，其与存在于5' UTR中的反式激活应答元件TAR结合。达特可与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，通过调节向生产性延伸的转变来控制转录的延伸阶段。P-TEFb本身由细胞RNA结合蛋白HEXIM 1或HEXIM 2与小核RNA 7SK的可逆结合调节。与游离形式的P-TEFb相比，这种大形式的P-TEFb相对无活性，但在许多细胞类型中代表P-TEFb的主要形式。因此，大部分潜在的P-TEFb活性保持在这个大的复合物中。最初的达特反式激活模型强调P-TEFb向新生病毒转录本的募集;然而，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。我们计划使用生物化学和结构生物学技术的组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合作努力中，现有的体外测定将用于寻找产生含有蛋白质或蛋白质和RNA的相关复合物的条件，这些复合物将形成晶体。最后，这些配合物的结构将使用晶体学技术解决。这些研究的结果将最终产生合理设计靶向HIV的疗法所需的结构和功能信息，并将为进一步了解P-TEFb对细胞转录的控制提供基础。本项目旨在了解HIV如何利用细胞P-TEFb调控机制来控制自身的转录程序，并为开发合理的HIV治疗方法提供前景。由病毒和细胞蛋白质和RNA组成的复合物将被结晶并确定结构。

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.