MOLECULAR ANALYSIS OF HSV-I REACTIVATION FROM LATENCY

拟议研究的长期目标是确定分子 潜伏性单纯疱疹向溶解性单纯疱疹转变的机制 病毒（HSV）基因转录。他们以前开发了高温 应力（HS）再激活模型，这是唯一的，在生产 在诱导后12-14小时内可检测到感染性病毒 刺激。使用这个模型，他们已经确定了可能是一个 关键事件在调控的重新激活，即上调 HS后1小时内的ICPO。 深入了解再激活的分子调控最终必须 通过分析单个潜伏感染的神经元获得。他们有 开发了一种新的方法，上下文表达式分析，CXA，以获得 细胞内单个细胞中DNA和RNA的定量信息 固体组织在本提案中，PCR和RT PCR的能力将是 通过CXA构建潜伏期的分子定义， 重新激活他们能够精确地量化潜在的 感染神经节中的神经元，并检查RNA和DNA含量， 使他们能够有意义地评估野生型和基因工程 突变菌株，以实现以下具体目的：（1）确定 潜伏感染的神经元的数量和/或 病毒基因组拷贝在个体潜伏感染的神经元内和/或 单个潜伏感染神经元内的病毒基因组拷贝数 在HS诱导的体内再活化的开始和进展时; (2)利用CXA-RNA策略来表征病毒在 潜伏期和以下HS诱导的反应在神经元群体和 单细胞水平;（3）确定生物学意义， HS后ICPO基因快速上调的生化基础 体内诱导反应。界定监管机制， 病毒遗传信息的“潜在”储存库周期性地引起 传染性病毒是理解这一重要方面的核心， 病毒生命周期对这些病毒功能的深入了解可能有助于 这对我们设计有效疫苗、开发 预防复发性疾病的治疗，并有效 在人类宿主中转移、维持和调节外源基因。

The long term goal of the proposed research is to define the molecular mechanisms involved in the transition from latent to lytic herpes simplex virus (HSV) gene transcription. They previously developed the hyperthermic stress (HS) reactivation model which is unique in that the production of infectious virus is detectable within 12-14 hours after the induction stimulus. Using this model, they have identified what is likely to be a key event in the regulation of reactivation, namely the up regulation of ICPO within 1 hour post HS. Insight into the molecular regulation of reactivation must ultimately be obtained from analysis of individual latently infected neurons. They have developed a new method, contextual expression analysis, CXA, to obtain quantitative information about the DNA and RNA in individual cells within solid tissues. In this proposal, the power of PCR and RT PCR will be harnessed through CXA to construct a molecular definition of latency and reactivation. Their ability to precisely quantify the number of latently infected neurons in the ganglia and examine the RNA and DNA content will allow them to meaningfully evaluate wild type and genetically engineered mutant strains to achieve the following specific aims: (1) Determine the impact of the number of latently infected neurons and/or the number of viral genome copies within individual latently infected neurons and/or the number of viral genome copies within individual latently infected neurons upon the initiation and progression of HS inducted reactivation in vivo; (2) Utilize CXA-RNA strategies to characterize viral transcription during latency and following HS induced reaction at the neuronal population and single cell level; (3) Determine the biological significance and biochemical basis of the rapid up regulation of the ICPO gene following HS induced reaction in vivo. Defining the regulatory mechanisms by which the "latent" repository of viral genetic information periodically give rise to infectious virus is central to understanding this important aspect of the viral life cycle. Insight into these viral functions could contribute significantly toward our ability to design effective vaccines, develop treatments for the prevention of recurrent disease, and efficiently transfer, maintain and regulate foreign genes in the human host.