Mechanisms by which VZV IE proteins interact with cell antiviral proteins to prev

描述（由申请人提供）： 项目总结/摘要：该提案描述了一个为期5年的研究和培训计划，玛丽亚内格尔博士发展一个独立的学术生涯研究水痘带状疱疹病毒（VZV）感染，潜伏期和再激活。超过90%的人群在青春期前感染VZV（Seward et al.，2000年）。由于老年人和免疫功能低下的个体对VZV的细胞介导的免疫力下降（Gershon和Steinberg，1981; Burke等人，1982年），VZV重新激活并导致多种严重的神经系统疾病，包括带状疱疹（带状疱疹），经常并发慢性疼痛（带状疱疹后神经痛），中风，失明和脊髓疾病。VZV在所有人类细胞中复制，除了病毒潜伏的神经元。决定生产性感染与潜伏性感染的机制尚不清楚，但可能涉及病毒蛋白和宿主细胞抗病毒防御机制的细胞类型特异性相互作用。最近对疱疹病毒的研究表明，病毒蛋白与宿主中的抗病毒早幼粒细胞白血病核体（PML-NB）相互作用，相互作用的结果有助于生产性感染而不是潜伏感染。（埃弗雷特和Chelbi-Alix，2007）;此外，与PML-NB在有丝分裂后细胞如神经元中的稳定性相比，PML-NB的结构在经历有丝分裂的细胞中显著改变（Dellaire等人，2006年a）。基于这些观察结果，我们假设VZV感染的结果（裂解与潜伏期和随后的再活化）是由VZV蛋白与宿主细胞的抗病毒PML-NB的细胞类型特异性相互作用引起的。为了验证这个假设，我们将：（目的1）分析用VZV有效感染的非神经元细胞的PML-NB与VZV基因61蛋白（VZV 61 p）的相互作用，所述VZV基因61蛋白与已知与PML-NB相互作用的疱疹病毒蛋白同源，以及与VZV基因63蛋白（VZV 63 p）的相互作用，所述VZV基因63蛋白是在潜伏感染的人神经节中表达的最普遍和丰富的VZV蛋白，用免疫化学和共聚焦显微镜。（目的2）使用目的1中的技术和荧光原位杂交检测神经节神经元中VZV DNA，分析在尸检中获得的潜伏感染的人神经节神经元中PML-NB与VZV 61 p和VZV 63 p的相互作用;和（目的3）使用来自目的2的技术分析在早期的神经节诱导的再活化期间在尸检中获得的人神经节神经元的PML-NB与VZV 61 p和VZV 63 p的相互作用。在获奖期间，内格尔博士将被晋升为神经病学系的助理教授，该系为医生-科学家提供了一个富有成效的环境。将通过分子病毒学、生物伦理学和统计分析方面的教学课程加强培训。她将把80%的精力用于研究，20%用于患者护理/临床教学。她将在培训的第四年申请独立的R 01奖。她的主要科学导师将是科罗拉多丹佛大学神经病学研究教授Randall Cohrs博士，他是一位基础研究科学家，也是世界上VZV发病机制的专家之一。尽管开发了带状疱疹疫苗，即使每个60岁以上的美国人都接种了带状疱疹疫苗，每年也会发生超过50万例带状疱疹新病例。因此，了解病毒和宿主细胞的相互作用，导致VZV的潜伏期和再激活，将提供战略的基础，以防止严重的神经系统疾病引起的VZV。 公共卫生相关性： 由嗜神经病毒引起的神经系统疾病是世界范围内发病率和死亡率的主要原因。水痘带状疱疹病毒比任何其他嗜神经病毒引起更多的神经系统疾病，但VZV和宿主细胞之间的相互作用，决定病毒是否建立生产与潜伏感染在很大程度上是未知的。更好地理解这些相互作用以及它们如何导致疾病是合理设计预防和治疗VZV和其他疱疹病毒引起的神经系统疾病的策略的先决条件。

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT: This proposal describes a 5-year research and training program for Dr. Maria Nagel to develop an independent academic career studying varicella zoster virus (VZV) infection, latency and reactivation. More than 90% of the population is infected with VZV before adolescence (Seward et al., 2000). As cell-mediated immunity to VZV declines in elderly and immunocompromised individuals (Gershon and Steinberg, 1981; Burke et al., 1982), VZV reactivates and causes multiple serious neurological diseases including zoster (shingles) frequently complicated by chronic pain (postherpetic neuralgia), stroke, blindness and spinal cord disease. VZV replicates in all human cells except neurons where virus becomes latent. The mechanisms that determine productive versus latent infection are unclear, but likely involve cell type-specific interactions of viral proteins and host cell antiviral defense mechanisms. Recent studies on herpesviruses show that viral proteins interact with antiviral promyelocytic leukemia nuclear bodies (PML-NBs) in the host and that the outcome of the interaction contributes to productive infection versus latency (Everett and Chelbi-Alix, 2007); in addition, the structure of PML-NBs is dramatically altered in cells undergoing mitosis compared to PML-NB stability in post-mitotic cells such as neurons (Dellaire et al., 2006a). Based on these observations, we hypothesize that the outcome of VZV infection (lysis versus latency and subsequent reactivation) results from cell type-specific interactions of VZV proteins with the host cell's antiviral PML-NBs. To test this hypothesis, we will: (Aim 1) analyze non-neuronal cells productively-infected with VZV for interactions of PML-NBs with VZV gene 61 protein (VZV 61p), which is homologous to herpesvirus proteins known to interact with PML-NBs, and with VZV gene 63 protein (VZV 63p), the most prevalent and abundant VZV protein expressed in latently-infected human ganglia, using immunochemistry and confocal microscopy. The effects of PML, VZV 61p, and VZV 63p overexpression and underexpression on VZV gene expression and viral titers will also be examined; (Aim 2) analyze latently-infected human ganglionic neurons obtained at autopsy for interactions of PML-NBs with VZV 61p and VZV 63p using techniques in Aim 1 and fluorescent in situ hybridization to detect VZV DNA in ganglionic neurons; and (Aim 3) analyze human ganglion neurons obtained at autopsy during early explant-induced reactivation for interactions of PML-NBs with VZV61p and VZV 63p using techniques from Aim 2. During the award period, Dr. Nagel will be promoted to Assistant Professor in the Department of Neurology, which provides a fruitful environment for physician-scientists. Training will be enhanced with didactic courses in molecular virology, bioethics, and statistical analysis. She will devote 80% effort to research and 20% to patient care/clinical teaching. She will apply for an independent R01 award during her 4th year of training. Her primary scientific mentor will be Dr. Randall Cohrs, Research Professor of Neurology at the University of Colorado Denver, a basic research scientist and one of the world's experts on VZV pathogenesis. Despite the development of a zoster vaccine, even if every American over age 60 received zoster vaccine, more than 500,000 new cases of zoster will occur annually. Thus, an understanding of viral and host cell interactions leading to VZV latency and reactivation will provide the basis for strategies to prevent serious neurological disease caused by VZV. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE: Neurological diseases caused by neurotropic viruses are a major cause of morbidity and mortality worldwide. Varicella zoster virus causes more neurological disease than any other neurotropic virus, yet the interactions between VZV and the host cell that determine whether the virus establishes productive versus latent infection are largely unknown. A better understanding of these interactions and how they lead to disease is prerequisite in the rational design of strategies to prevent and treat neurological disease produced by VZV and other herpesviruses.