Regulation of P-TEFb during HIV Infection

描述（由申请人提供）：HIV需要一种细胞蛋白P-TEFb的功能，该蛋白允许产生全长HIV转录物。病毒合成蛋白质达特，其与P-TEFb缔合并通过达特与新生病毒转录物的相互作用将其带到病毒转录单位。 P-TEFb是一种细胞周期蛋白依赖性激酶，由Cdk 9和几种可能的细胞周期蛋白亚基之一组成，达特仅募集含有细胞周期蛋白TI的P-TEFb。已经发现了许多抑制P-TEFb的小化合物，并且因为P-TEFb是大多数细胞基因表达所需的，所以在高浓度下这些抑制剂导致细胞死亡。在低得多的浓度下，所有P-TEFb抑制剂都阻断HIV复制，而对正常细胞功能没有影响。这种增强的灵敏度的原因尚不清楚，但我们的假设是P-TEFb相关分子负责。为了解决这个问题，我们提出了一个详细的检查量，亚细胞位置和功能的P-TEFb组件在常用的细胞系和主要人体组织相关的HIV感染。这些成分包括Cdk 9，一种新发现的Cdkg的替代形式，cyclinT 1和7SK，一种似乎参与控制P-TEFb活性的小细胞RNA。生物化学研究将研究P-TEFb激酶活性的功能及其在转录中的作用，重点是为什么HIV基因的表达对P-TEFb抑制比正常细胞基因更敏感。重要的是，将使用HeLa和Jurkat细胞系以及原代人单核细胞衍生的巨噬细胞和外周血淋巴细胞进行各种HIV感染研究。总之，这些研究不仅将增强我们对达特反式激活机制的理解，而且还将使我们能够确定HIV感染如何改变细胞的P-TEFb环境。最后，我们的研究结果也将有助于评估有效的P-TEFb抑制剂，flavopiridol，作为一种抗HIV治疗。

DESCRIPTION (provided by applicant): HIV requires the function of a cellular protein, P-TEFb, that allows full length HIV transcripts to be produced. The virus synthesizes a protein, Tat, that associates with P-TEFb and brings it to the viral transcription unit through an interaction of Tat with the nascent viral transcript. P-TEFb is a cyclin dependent kinase comprised of Cdk9 and one of several possible cyclin subunits and Tat recruits only P-TEFb containing cyclinTI. A number of small compounds have been found that inhibit P-TEFb and because P-TEFb is required for the expression of most cellular genes, at high concentrations these inhibitors cause cell death. At much lower concentrations all P-TEFb inhibitors block HIV replication while having no effect on normal cellular function. The reason for this enhanced sensitivity is not known, but it is our hypothesis that P-TEFb associated molecules are responsible. To address this issue,we propose a detailed examination of the amount, subcellular location and function of P-TEFb components in commonly used cell lines and in primary human tissues relevant to HIV infection. These components include Cdk9, a newly discovered alternative form of Cdkg, cyclinT1, and 7SK, a small cellular RNA that seems to be involved in controlling the activity of P-TEFb. Biochemical studies will investigate the function of P-TEFb kinase activity and its role in transcription with an emphasis on why the expression of HIV genes is so much more sensitive to P-TEFb inhibition than normal cellular genes. Importantly, a variety of HIV infection studies will be carried out using HeLa and Jurkat cell lines and primary human monocyte derived macrophages and peripheral blood lymphocytes. In total, these studies will not only enhance our understanding of the mechanism of Tat transactivation, but will also allow us to determine how HIV infection may alter the P-TEFb environment of a cell. Finally, our results will also be useful in evaluating the potent P-TEFb inhibitor, flavopiridol, as an anti-HIV therapy.