Molecular mechanisms regulating HIV entry and exit from latency

描述(申请人提供)：感染细胞的能力潜伏地帮助艾滋病毒建立持续感染，尽管对病毒蛋白有强大的体液和细胞免疫反应，并且仍然是开发治疗方法的最大障碍，从而导致从感染者身上消除艾滋病毒。这一建议建立在我们最近进行的一些引人注目的观察的基础上，这些观察表明艾滋病毒转录的激活是一个非常动态的过程。我们利用高分辨率染色质免疫沉淀(CHIP)实验发现，随着NF-kappaB进入和离开细胞核，RNA聚合酶、TFIIH和许多其他转录机器组件对前病毒启动子的招募迅速上升和下降。HIV对细胞激活信号的转录反应是间歇性的，而不仅仅是对初始信号事件的不可逆反应，这一发现完全出乎人们的意料。在补充性研究中，我们还计划调查HIV进入潜伏期的分子机制。我们最近发现，在顺式病毒中携带TAT的整合慢病毒载体的转录在4到6周内逐渐减少，直到病毒进入潜伏状态。转录的关闭似乎与TAT活性的丧失有关，因为降低其活性的TAT突变会导致更快的病毒关闭。这些观察结果导致了一种假设，即一旦启动率下降到不再产生TAT阈值水平时，艾滋病毒就进入潜伏状态。核因子-kappaB和相关转录因子的激活恢复了转录启动，并重新建立了TAT反馈机制，从而导致病毒RNA的高水平合成。由于HIV转录对细胞刺激的重新激活是一个高度动态的过程，在这个过程中，多个转录因子在启动子上迅速招募和交换，因此可以使用多个信号通路来调节HIV对外部刺激的转录反应的时间和幅度。我们计划结合模型细胞系统中HIV启动子激活的生化研究，以及针对前病毒静止和潜伏期的初级细胞模型的开发，严格检验这一假说。这些研究有望带来对驱动艾滋病毒潜伏期的具体因素的详细新理解，并为抗病毒研究提供新的途径。

DESCRIPTION (provided by applicant): The ability to infect cells latently helps HIV to establish persistent infections despite strong humoral and cellular immune responses against the viral proteins and remains the single greatest obstacle to the development of therapies that result in the elimination of HIV from infected individuals. This proposal builds on some remarkable observations we have recently made demonstrating that the activation of HIV transcription is an extraordinarily dynamic process. We have found using high resolution chromatin immunoprecipitation (ChIP) experiments that the recruitment of RNA polymerase; TFIIH and numerous other components of the transcription machinery to the proviral promoter rapidly rises and falls as NF-kappaB enters and exits the nucleus. The discovery that the transcriptional responses of HIV to cellular activation signals is intermittent, and not simply an irreversible response to an initial signaling event, was completely unexpected. In complementary studies, we also plan to investigate the molecular mechanisms utilized by HIV to enter latency. We have recently found that transcription from integrated lentiviral vectors that carry Tat in cis progressively diminishes over a period of 4 to 6 weeks until the virus enters a latent state. The shutdown in transcription appears to be associated with a loss of Tat activity since mutations in Tat that reduce its activity lead to more rapid viral shutdown. These observations lead to the hypothesis that HIV enters a latent state once initiation rates have fallen to a level where threshold levels of Tat are no longer produced. Activation of NF-kappaB and related transcription factors restores transcription initiation and re-establishes the Tat feedback mechanism leading to high levels of viral RNA synthesis. Because reactivation of HIV transcription in response to cellular stimulation is a highly dynamic process in which multiple transcription factors are rapidly recruited and exchanged at the promoter, multiple signaling pathways can be used to regulate both the timing and magnitude of the transcriptional response of HIV to external stimuli. We plan to rigorously test this hypothesis using a combination of biochemical studies of HIV promoter activation in model cellular systems, and the development of primary cell models for proviral quiescence and latency. These studies promise to lead to a detailed new understanding of the specific factors that drive HIV latency and suggest new avenues for antiviral research.