Role of Factor Acetylation in the Regulation of HIV Transcription

描述（由申请人提供）：转录是HIV-1生命周期中的关键调控步骤，在很大程度上尚未作为治疗靶点进行探索。我们已经确定因子乙酰化是调节HIV-1转录的关键步骤。乙酰化是乙酰基（14 Da）可逆转移到赖氨酸的-氨基。它由乙酰转移酶（HAT）介导，并由脱乙酰酶（HDAC）的活性逆转。HDAC抑制剂目前正在探索作为潜在的治疗剂，以冲洗HIV感染患者的潜伏水库。HIV转录的调节中有三个因素：达特、NF-B和细胞周期蛋白T1/P-TEFb。值得注意的是，达特和NF-B的功能受到这些蛋白质可逆乙酰化的重要调节。我们现在报告，P-TEFb中的细胞周期蛋白T1也在四个定义的乙酰受体位点乙酰化。细胞周期蛋白T1乙酰化通过促进P-TEFb从含有7SK RNA和Hexim 1的核糖核蛋白复合物中解离来激活P-TEFb，并重要地调节HIV LTR的活性。此外，我们已经确定了一种新的翻译后修饰达特，单甲基化赖氨酸51的Set 9，调节达特乙酰化和激活达特转录活性。我们建议研究这两个新发现对HIV转录过程的影响。我们推测，可逆的细胞周期蛋白T1乙酰化是一个重要的调节机制，控制活动和潜伏期的HIV感染的HIV启动子的活性。此外，我们推测达特单甲基化是促进达特乙酰化从而激活达特转录活性的关键步骤。我们的具体目标是测试这些假设，并确定与细胞周期蛋白T1乙酰化和达特甲基化/乙酰化在活跃或潜伏性HIV感染的T细胞的机制。我们的重点是1。评估cyclinT 1乙酰化在HIV复制中的作用。我们建议检查是否乙酰化缺陷的突变体细胞周期蛋白T1可以支持HIV复制的CD 4 + T细胞和细胞周期蛋白T1乙酰化是否调节建立和维持后整合潜伏期。我们的初步研究结果表明，细胞周期蛋白T1乙酰化是一个重要的调节机制的活动的HIV LTR，而它可能不影响达特反式激活。由于HIV LTR的基础活性在感染过程开始时或在达特蛋白尚未产生时从潜伏期重新激活期间是至关重要的，因此我们推测细胞周期蛋白T1乙酰化可能是HIV在这些时间转录的关键决定因素。2.表征细胞周期蛋白T1乙酰化如何调节HIV启动子处的P-TEFb功能。我们建议测试的假设，乙酰化的赖氨酸在细胞周期蛋白T1与溴结构域Brd 4的相互作用。Brd 4是在不存在达特的情况下可以将P-TEFb募集到HIV启动子的因子，并且含有两个溴结构域，其是乙酰基赖氨酸的真正结合结构域。我们推测细胞周期蛋白T1乙酰化通过将Brd 4募集到活性P-TEF复合物中来激活HIV LTR活性。同样，我们将研究乙酰化的细胞周期蛋白T1如何与达特和TAR RNA相互作用。3.研究赖氨酸甲基化如何与达特乙酰化协同调控HIV转录。我们建议研究Set 9的达特单甲基化和p300的达特乙酰化之间的潜在串扰。我们的初步数据表明，达特单甲基化是达特反式激活循环的早期步骤，先于达特乙酰化。我们将测试单甲基化达特或Set 9是否将p300募集到达特。我们还将使用我实验室产生的修饰特异性达特抗体，研究不同修饰的达特种类如何在HIV-1感染的T细胞中积累。我们预计这些研究将增加我们对HIV转录生物学的分子理解，并为HDAC抑制剂在HIV感染中的治疗潜力提供新的见解。公共卫生相关性：我们试图确定和表征控制HIV-1转录的新的调控机制，这些机制可能被用作新的治疗靶点。我们提出的研究特点的作用，可逆的因子乙酰化（细胞周期蛋白T1和达特）在控制活动和潜伏的HIV感染。这些研究与艾滋病毒/艾滋病直接相关，可能有助于开发新的抗病毒药物，以满足公众的需求。

DESCRIPTION (provided by applicant): Transcription is a key regulatory step in the HIV-1 life cycle that remains largely unexplored as therapeutic target. We have identified factor acetylation as a critical step in the regulation of HIV-1 transcription. Acetylation is the reversible transfer of an acetyl group (14 Da) to the -amino group of lysines. It is mediated by acetyl transferases (HAT) and reversed by the activity of deacetylases (HDACs). Inhibitors of HDACs are currently being explored as potential therapeutics to flush the latent reservoir in HIV-infected patients. Three factors stand out in the regulation of HIV transcription: Tat, NF-B and cyclin T1/P-TEFb. Notably, the functions of Tat and NF-B are importantly regulated by reversible acetylation of these proteins. We now report that cyclin T1 in P-TEFb is also acetylated at four defined acetyl acceptor sites. Cyclin T1 acetylation activates P-TEFb by promoting its dissociation from ribonucleoprotein complexes containing 7SK RNA and Hexim1 and importantly regulates the activity of the HIV LTR. In addition, we have identified a novel posttranslational modification in Tat, monomethylation of lysine 51 by Set9 that regulates Tat acetylation and activates Tat transcriptional activity. We propose to examine the impact of these two new findings on the HIV transcriptional process. We hypothesize that reversible cyclin T1 acetylation is an important regulatory mechanism that controls the activity of the HIV promoter during active and latent HIV infection. In addition, we speculate that Tat monomethylation is a critical step to promote Tat acetylation thereby activating Tat transcriptional activity. Our specific aims are designed to test these hypotheses and to define the mechanisms associated with cyclin T1 acetylation and Tat methylation/acetylation in actively or latently HIV-infected T cells. Our focus lies on 1. Assessing the role of cyclinT1 acetylation in HIV replication. We propose to examine whether acetylation- deficient mutants of cyclinT1 can support HIV replication in CD4+ T cells and whether cyclin T1 acetylation regulates the establishment and maintenance of post-integration latency. Our preliminary results show that cyclin T1 acetylation is an important regulatory mechanism for the activity of the HIV LTR while it may not affect Tat transactivation. Since the basal activity of the HIV LTR is critical at the beginning of the infectious process or during reactivation from latency when the Tat protein is not yet produced, we speculate that cyclin T1 acetylation might be a critical determinant of HIV transcription at these times. 2. Characterizing how cyclin T1 acetylation regulates P-TEFb function at the HIV promoter. We propose to test the hypothesis that acetylated lysines in cyclin T1 interact with bromodomains in Brd4. Brd4 is a factor that can recruit P-TEFb to the HIV promoter in the absence of Tat and contains two bromodomains, which are bona fide binding domains for acetyl lysines. We speculate that cyclin T1 acetylation activates HIV LTR activity by recruiting Brd4 into the active P-TEF complex. Similarly, we will examine how acetylated cyclin T1 interacts with Tat and TAR RNA. 3. Studying how lysine methylation cooperates with Tat acetylation to regulate HIV transcription. We propose to study the potential crosstalk between Tat monomethylation by Set9 and Tat acetylation by p300. Our preliminary data show that Tat monomethylation is an early step in the Tat transactivation cycle and precedes Tat acetylation. We will test whether monomethylated Tat or Set9 recruits p300 to Tat. We will also examine how differently modified Tat species accumulate in HIV-1-infected T cells using modification-specific Tat antibodies generated in my laboratory. We anticipate that these studies will increase our molecular understanding of the biology of HIV transcription and provide novel insight into the therapeutic potential of HDAC inhibitors in HIV infection. PUBLIC HEALTH RELEVANCE: We seek to identify and characterize novel regulatory mechanisms controlling HIV-1 transcription that might be exploited as new therapeutic targets. Our proposed studies characterize the role of reversible factor acetylation (cyclin T1 and Tat) in the control of active and latent HIV infection. These studies are directly relevant to HIV/AIDS and may contribute to the development of novel antiviral drugs that will address public need.