Regulation of P-TEFb during HIV Infection

HIV 感染期间 P-TEFb 的调节

• 批准号: 6927571
• 负责人: David H Price
• 金额: $ 2.99万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6927571
• 关键词: HIV infections; T lymphocyte; charge coupled device camera; clinical research; cyclin dependent kinase; flavopiridol; gene expression; genetic transcription; human immunodeficiency virus; human tissue; immunocytochemistry; immunoprecipitation; immunoregulation; in situ hybridization; macrophage; northern blottings; protein localization; tissue /cell culture; virus replication; western blottings

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.

描述（由申请人提供）：HIV需要细胞蛋白P-TEFB的功能，该功能允许产生全长HIV转录本。该病毒合成了与P-TEFB相关的蛋白质TAT，并通过TAT与新生病毒转录本的相互作用将其带入病毒转录单元。 P-TEFB是一种依赖于CDK9的细胞周期蛋白激酶，也是几个可能的细胞周期蛋白亚基之一，而TAT菌株仅含有cyclinti的P-TEFB。已经发现许多小型化合物抑制P-TEFB，并且由于表达大多数细胞基因所必需的P-TEFB，因此在高浓度下这些抑制剂会导致细胞死亡。在较低的浓度下，所有P-TEFB抑制剂都会阻止HIV复制，同时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相关的分子是负责的。为了解决这个问题，我们提出了对常用细胞系中P-TEFB成分的数量，亚细胞位置和功能的详细检查以及与HIV感染相关的原发性人体组织中的量。这些组件包括CDK9，这是一种新发现的CDKG，Cyclint1和7sk的替代形式，这是一种小细胞RNA，似乎与控制P-TEFB活性有关。生化研究将研究P-TEFB激酶活性的功能及其在转录中的作用，强调为什么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多种HIV感染研究。总的来说，这些研究不仅将增强我们对TAT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，我们的结果也将有助于评估有效的P-TEFB抑制剂黄酮醇作为抗HIV疗法。