Molecular mechanisms regulating HIV entry and exit from latency

描述（由申请方提供）：尽管针对病毒蛋白的体液和细胞免疫应答很强，但潜伏感染细胞的能力有助于HIV建立持续感染，并且仍然是开发导致从感染个体中消除HIV的疗法的最大障碍。这个建议是建立在我们最近的一些显著观察基础上的，这些观察表明HIV转录的激活是一个非常动态的过程。我们已经发现使用高分辨率染色质免疫沉淀（ChIP）实验，RNA聚合酶、TFIIH和转录机制的许多其他组分向前病毒启动子的募集随着NF-κ B进入和离开细胞核而迅速上升和福尔斯下降。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，这一发现完全出乎意料。在补充研究中，我们还计划研究HIV进入潜伏期的分子机制。我们最近发现，从顺式携带达特的整合慢病毒载体的转录在4至6周的时间内逐渐减少，直到病毒进入潜伏状态。转录的关闭似乎与达特活性的丧失有关，因为降低其活性的达特突变导致更快的病毒关闭。这些观察结果导致一种假设，即一旦启动率下降到不再产生达特阈值水平的水平，艾滋病毒就进入潜伏状态。NF-kappaB和相关转录因子的激活恢复转录起始并重新建立达特反馈机制，导致高水平的病毒RNA合成。因为响应于细胞刺激的HIV转录的再激活是一个高度动态的过程，其中多种转录因子在启动子处被快速募集和交换，所以可以使用多种信号传导途径来调节HIV对外部刺激的转录响应的时间和幅度。我们计划使用模型细胞系统中HIV启动子激活的生物化学研究和原病毒静止和潜伏期的原代细胞模型的开发相结合来严格检验这一假设。这些研究有望导致对驱动HIV潜伏期的特定因素的详细新理解，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.