CYCLIN T AND HIV1 TAT TRANSACTIVATION

我们最近分离出一种新的c型细胞周期蛋白（称为细胞周期蛋白T），它通过其激活域直接与HIV-1 Tat相互作用。细胞周期蛋白T （CycT）是CDK9 （PITALRE）的主要细胞周期蛋白伴侣，CDK9是正作用转录延伸因子复合物P-TEFb的催化亚基。我们确定Tat与CycT的相互作用显著增强了其与TAR RNA的结合，并赋予了TAR环中不被游离（未络合）Tat蛋白识别的关键序列的要求。周期蛋白T:Tat相互作用的结构-功能分析。在这里，我们建议在分子细节上表征Tat和CycT之间的相互作用，并确定Tat相关激酶(TAK)/P-TEFb复合体中存在的其他因子，这些因子可能是Tat调控转录延伸所必需的。在Specific Aim 1中，我们将使用定点诱变和UV交联技术鉴定CycT和Tat中相互作用和与Tar RNA结合所必需的残基。在Specific Aim 2中，我们将分析野生型和突变型CycT蛋白在体内支持基础和Tat介导的转激活的能力，以及在hCycT免疫缺失或被反式显性Tat（1-48）蛋白阻断的无细胞转录反应中的能力。我们还将确定Tat活性是否需要额外的P-TEFb成分。我们最近克隆了小鼠CycT蛋白（mCycT），发现它不能通过TAR支持Tat的转激活。在Specific Aim 3中，我们将描述mCycT中的缺陷，并确定在体内恢复Tat转激活所需的最小变化。最后，在Specific Aims 4和5中，我们提出克隆和表征两个新的蛋白，我们发现它们在核提取物中与TAK/P- TEFb紧密相关，无论是单独的还是与TAR RNA结合的。综上所述，这些研究将提供有关HIV Tat转录激活机制的重要新信息。

We have recently isolated a novel C-type cyclin (called Cyclin T) which interacts directly with HIV-1 Tat through its activation domain. Cyclin T (CycT) is a predominant cyclin partner for CDK9 (PITALRE), the catalytic subunit of the positive-acting transcription elongation factor complex, P-TEFb. We determined that the interaction of Tat with CycT dramatically enhances its binding to TAR RNA, and confers a requirement for critical sequences in the loop of TAR which are not recognized by the free (uncomplexed) Tat protein. Structure-function analysis of the cyclin T:Tat interaction. Here we propose to characterize the interaction between Tat and CycT in molecular detail, and to identify other factors present in the Tat-associated kinase (TAK)/P-TEFb complex that may be required for the regulation of transcription elongation by Tat. In Specific Aim 1 we will identify residues within CycT and in Tat that are necessary for their interaction and for binding to Tar RNA using site-directed mutagenesis and UV cross-linking techniques. In Specific Aim 2, we will analyze the ability of wild-type and mutant CycT proteins to support basal and Tat-mediated transactivation in vivo and in cell-free transcription reactions that have been immunodepleted of hCycT, or blocked with trans-dominant Tat (1-48) protein. We will also determine whether additional P-TEFb components are needed for Tat activity. We have recently cloned the murine CycT protein (mCycT) and find that it is unable to support Tat transactivation through TAR. In Specific Aim 3, we will characterize the defect in mCycT and identify the minimal changes needed to restore Tat transactivation in vivo. Finally, in Specific Aims 4 and 5 we propose to clone and characterize two novel proteins which we find to be tightly associated with TAK/P- TEFb in nuclear extracts, either alone or when bound to TAR RNA. Taken together, these studies will provide important new information on the mechanism of HIV Tat transactivation.