Molecular Analysis of HSV-1 Reactivation from Latency

描述(申请人提供)：单纯疱疹病毒(HSV)感染在全球人口中继续流行，造成严重的健康后果。干预从潜伏状态重新激活单纯疱疹病毒是努力控制这一流行病的关键组成部分。这项研究的长期目标是获得控制从潜伏期转录到裂解期转录的分子机制的知识，用于合理设计干预策略。人们普遍认为，病毒/宿主细胞的相互作用在初次感染期间启动病毒裂解级联，与从潜伏的病毒基因组启动裂解级联不同。这一概念植根于这样一个事实，即α基因转基因诱导因子(VP16)是作为蛋白质与病毒粒子一起进入的，直到裂解周期的后期，当它作为蛋白质被结合到病毒粒子中时才产生。因此，这种重要的病毒反式激活因子被认为在潜伏期和潜伏的病毒基因组转录激活期间都不存在。这就提出了确定病毒/宿主转录激活界面的必要性，而不是明确定义的VP16/Octl/HCF/taatgarat复合体。尽管IE基因ICP0被认为有可能在启动裂解周期中“取代”VP16，而且对ICP0功能的研究揭示了该蛋白在改变宿主细胞环境中的作用，但仍没有证据表明ICP0启动进入裂解周期。我们已经产生了令人信服的数据，无论是表型数据还是生化数据，都标志着VP16在启动重新激活过程中发挥了关键作用。这一关键观察需要重点研究病毒IE基因转录从潜伏期上调的机制。以下具体目标将指导我们系统地剖析(1)VP16在启动再激活过程中的作用；(2)VP16在应激驱动上调所需的启动子元件；(3)病毒基因组结构在调节转录沉默和启动再激活中的作用。为了便于对神经节中单个神经元中发生的事件进行常规检查和量化，开发和改进方法是我们取得进展的基础。这些分析工具以及病毒基因组的精确工程和染色质结构分析的进展将被用于这些研究。

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) infection continues at epidemic levels in the global human population with serious health consequences. Intervention of the reactivation of HSV from the latent state is a critical component of efforts to control this epidemic. The long-term goal of the proposed research is to acquire knowledge of the molecular mechanisms controlling the transition from latent to lytic phase transcription for use in the rational design of intervention strategies. It is widely accepted that the viral/host cell interactions initiating the viral lytic cascade during primary infection are distinct from those initiating the lytic cascade from the latent viral genome. This notion is rooted in the fact that the alpha gene transinducing factor (VP16) is brought in with the virion as a protein and not made until late in the lytic cycle when it is incorporated as protein into the virion. Thus this important viral transactivator is thought to be absent during latency as well as during activation of transcription from the latent viral genome. This presents the necessity to identify a viral/host transcriptional activation interface other than the well-defined VP16/Octl/HCF/taatgarat complex. Although the IE gene ICP0 has been viewed as the likely candidate to "replace" VP16 in initiating the lytic cycle, and studies of ICP0 function have revealed much about the role of this protein in modifying the host cell environment, there remains no evidence that ICP0 initiates entry into the lytic cycle. We have generated compelling data, both phenotypic and biochemical that marks VP16 as a critical player in the initiation of reactivation. This key observation calls for a focused examination of the mechanism of upregulation of viral IE gene transcription from latency. The following specific aims will guide us through a systematic dissection of (1) the role of VP16 during the initiation of reactivation; (2) the VP16 promoter elements required for its stress driven upregulation; and (3) the role of viral genome structure in the regulation of transcriptional silencing and initiation of reactivation. The development and refinement of methodologies to facilitate routine examination and quantification of the events occurring in individual neurons in the ganglia have been fundamental to our progress. These analytical tools together with precision engineering of the viral genome and advances in the analysis of chromatin structure will be utilized for these studies.