Molecular mechanisms of cellular gene expression reprogramming by HIV Tat protein

描述（由申请人提供）：本提案的目标是深入了解HIV-1 Tat如何介导其对宿主细胞基因表达的影响。它是HIV-1感染期间最先表达的基因之一，对病毒基因表达和病毒产生至关重要。它通过作为延伸因子的功能和与TAR（一种存在于HIV病毒转录物开始的RNA序列）以及宿主细胞因子CDK9和细胞周期蛋白T1相互作用，增加HIV-1基因的表达。Tat与这些和其他宿主细胞转录调节因子的相互作用可能会影响宿主细胞基因表达。事实上，有大量证据表明，Tat可以影响T淋巴细胞、神经元和抗原提呈细胞的生理机能。我们实验室最近的研究表明，树突状细胞和巨噬细胞的基因表达程序会因病原体暴露而改变，这些改变可以为其发病机制提供重要线索。我们已经证明，HIV-1介导的树突状细胞重编程可以创造有利于病毒传播的条件，并且这种效果是由HIV-1反激活子Tat介导的。这一建议旨在提高我们对Tat表达引起HIV-1靶向免疫细胞中宿主细胞基因表达变化的手段的理解。为此，本研究的具体目标是：1)建立表达多种野生型和突变Tat蛋白的腺病毒和逆转录病毒载体文库。2)研究Tat两种野生型等位基因对HIV-1感染靶向免疫细胞中宿主细胞基因表达的影响。3)绘制负责修饰宿主细胞基因表达程序的Tat结构域。4)研究转录因子STAT1和IRF7在Tat表达过程中的基因组定位，因为这些因子在HIV-1感染和Tat表达中上调。5)利用设计用于检测DNA和RNA结合的基因组尺度定位分析实验，研究Tat重编程宿主细胞基因表达的机制。

DESCRIPTION (provided by applicant): The goal of this proposal is to gain insights into how HIV-1 Tat mediates its effects on host cell gene expression. Tat is among the first genes expressed during HIV-1 infection and is essential for viral gene expression and virus production. Tat increases HIV-1 gene expression by functioning as an elongation factor and interacting with TAR, a RNA sequence present at the beginning of the HIV viral transcripts, and with the host cell factors CDK9 and cyclin T1. The interaction of Tat with these and other host cell transcriptional regulators might be expected to affect host cell gene expression. Indeed, there is considerable evidence that Tat can affect the physiology of T lymphocytes, neurons and antigen presenting cells. Recent studies from our laboratory have shown that the gene expression programs of dendritic cells and macrophages are modified by pathogen exposure and that these modifications can provide important clues to pathogenesis. We have shown that dendritic cell reprogramming by HIV-1 can create conditions that favor virus spread, and that the effect is mediated by the HIV-1 transactivator Tat. This proposal is designed to improve our understanding of the means by which Tat expression causes changes in host cell gene expression in immune cells targeted by HIV-1. To accomplish this, the specific aims of the proposal are 1) To develop a library of adenovirus and retroviral vectors expressing multiple wild type and mutated Tat proteins. 2) To determine the effects of two wild type alleles of Tat on host cell gene expression in immune cells targeted by HIV-1 infection. 3) To map the domain of Tat that is responsible for modifying the host cell gene expression program. 4) To investigate the genome-scale location of the transcription factors STAT1 and IRF7 during Tat expression, as these factors are upregulated by HIV-1 infection and Tat expression. 5) To investigate the mechanism by which Tat reprograms host cell gene expression using genome-scale location analysis experiments designed to detect DNA and RNA binding.