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

描述（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。一个小的病毒蛋白Tat和病毒转录物的5'未翻译区（UTR）都参与了这个过程。Tat是一种RNA结合蛋白，与存在于5' UTR中的反式激活反应元件TAR结合。它可以与TAR和P-TEFb形成络合物。P-TEFb是一种依赖于细胞周期蛋白的激酶，通过调节转录向生产性伸长的转变来控制转录的伸长阶段。P-TEFb本身受细胞RNA结合蛋白HEXIM1或HEXIM2与小核RNA 7SK的可逆结合调控。与游离形式的P-TEFb相比，这种大形式的P-TEFb相对不活跃，但在许多细胞类型中代表了P-TEFb的主要形式。因此，大部分潜在的P-TEFb活性都在这个大的复合物中。Tat交易激活的原始模型强调P-TEFb对新生病毒转录物的招募；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。我们计划结合生物化学和结构生物学技术来研究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合作中，现有的体外试验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用晶体学技术来解决。这些研究的结果将最终为合理设计针对HIV的治疗方法提供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。该项目旨在了解HIV如何利用细胞P-TEFb调控机制来控制其自身的转录程序，以期开发出合理的HIV治疗方法。由病毒和细胞蛋白质和rna组成的复合物将结晶并确定结构。

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.