MOLECULAR ANALYSIS OF HSV-I REACTIVATION FROM LATENCY

这项拟议研究的长期目标是定义分子 潜伏型单纯疱疹向融解型单纯疱疹转化的机制 病毒(HSV)基因转录。他们之前开发出了高温症 应激(HS)再激活模型，其独特之处在于产生 在诱导后12-14小时内可检测到传染性病毒 刺激。使用这种模型，他们已经确定了很可能是 再激活调控中的关键事件，即上调 ICPO在HS后1小时内。 对重新激活的分子调控的洞察最终必须 从对单个潜伏感染神经元的分析中获得。他们有 发展了一种新的方法，上下文表达式分析，CXA，以获得 关于单个细胞中DNA和RNA的定量信息 固体组织。在这项提案中，聚合酶链式反应和逆转录聚合酶链式反应的能力将是 通过CXA构建了潜伏期和 重新激活。他们能够精确量化潜伏着的 感染神经节中的神经元并检测RNA和DNA含量将 允许他们有意义地评估野生型和转基因 突变株达到以下具体目的：(1)测定 潜伏感染神经元的数量和/或 单个潜伏感染神经元内的病毒基因组复制和/或 单个潜伏感染神经元内的病毒基因组拷贝数 在活体内HS诱导再激活的启动和进展； (2)利用CXA-RNA策略来表征病毒在 潜伏期和紧随的HS引起的神经元群体和 单细胞水平；(3)确定生物学意义和 HS后ICPO基因快速上调的生化基础 在体内诱导反应。定义监管机制，以使 “潜伏”的病毒遗传信息库周期性地引起 传染性病毒是理解病毒的这一重要方面的核心 病毒的生命周期。对这些病毒功能的洞察可能有助于 对于我们设计有效疫苗的能力，开发 预防复发疾病的治疗，并有效 在人类宿主中转移、维持和调节外来基因。

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.