Role of Factor Acetylation in the Regulation of HIV Transcription

描述(由申请人提供)：转录是HIV-1生命周期中的一个关键调控步骤，作为治疗靶点在很大程度上仍未被探索。我们发现因子乙酰化是HIV-1转录调控的关键步骤。乙酰化是将一个乙酰基(14Da)可逆地转移到赖氨酸的氨基上。它由乙酰转移酶(HAT)介导，并由脱乙酰酶(HDACs)活性逆转。目前正在探索HDACs的抑制剂作为潜在的治疗药物来冲洗艾滋病毒感染患者的潜伏期。在HIV转录调控中有三个因子：TAT、NF-B和细胞周期蛋白T1/P-TEFb。值得注意的是，TAT和核因子-B的功能受这些蛋白质的可逆乙酰化的重要调节。我们现在报道，P-TEFb中的Cyclin T1也在四个定义的乙酰基受体位置发生乙酰化。Cyclin T1乙酰化通过促进P-TEFb从含有7SK RNA和HEXIM1的核糖核蛋白复合体中解离来激活P-TEFb，并重要地调节HIV LTR的活性。此外，我们还发现了TAT的一种新的翻译后修饰，即Set9对赖氨酸51的单甲基化，它调节TAT的乙酰化并激活TAT的转录活性。我们建议检查这两个新发现对艾滋病毒转录过程的影响。我们推测，可逆的细胞周期蛋白T1乙酰化是控制HIV启动子活性的重要调控机制。此外，我们推测TAT单甲基化是促进TAT乙酰化从而激活TAT转录活性的关键步骤。我们的具体目标是测试这些假说，并确定与主动或潜伏感染HIV的T细胞中细胞周期蛋白T1乙酰化和TAT甲基化/乙酰化相关的机制。我们的重点在于1.评估细胞周期蛋白T1乙酰化在HIV复制中的作用。我们建议检查是否Cyclin T1的乙酰化缺陷突变体可以支持HIV在CD4+T细胞中的复制，以及Cyclin T1乙酰化是否调节整合后潜伏期的建立和维持。我们的初步结果表明，Cyclin T1乙酰化是HIV LTR活性的一个重要调节机制，而它可能不影响TAT的反式激活。由于HIV LTR的基本活性在感染过程的开始或TAT蛋白尚未产生的潜伏期重新激活期间是关键的，我们推测Cyclin T1乙酰化可能是这些时候HIV转录的关键决定因素。2.研究Cyclin T1乙酰化如何调节HIV启动子上的P-TEFb功能。我们建议检验Cyclin T1中的乙酰化赖氨酸与Brd4中的溴结构域相互作用的假设。BRD4是一种在缺乏TAT的情况下可以将P-TEFb募集到HIV启动子上的因子，它含有两个溴区，它们是乙酰赖氨酸的真正结合结构域。我们推测，Cyclin T1乙酰化通过将Brd4招募到活性的P-TEF复合体中来激活HIV LTR活性。同样，我们将研究乙酰化细胞周期蛋白T1如何与TAT和TAR RNA相互作用。3.研究赖氨酸甲基化与TAT乙酰化如何协同调控HIV转录。我们建议研究TAT单甲基化和TAT乙酰化之间的潜在串扰。我们的初步数据显示，TAT单甲基化是TAT反式激活循环的早期步骤，先于TAT乙酰化。我们将测试单甲基化的TAT或Set9是否招募p300到TAT。我们还将使用我的实验室产生的修饰特异性TAT抗体，研究不同修饰的TAT物种如何在感染HIV-1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：我们寻求识别和表征控制HIV-1转录的新调控机制，这些机制可能被用作新的治疗靶点。我们的研究描述了可逆因子乙酰化(细胞周期蛋白T1和TAT)在控制HIV主动和潜伏感染中的作用。这些研究与艾滋病毒/艾滋病直接相关，并可能有助于开发满足公众需求的新型抗病毒药物。

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.