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

描述(由申请人提供)： 项目摘要/摘要：本提案描述了一项为期5年的研究和培训计划，该计划旨在让Maria Nagel博士发展独立的学术生涯，研究水痘带状疱疹病毒(VZV)的感染、潜伏和重新激活。超过90%的人口在青春期之前感染了VZV(Seward等人，2000年)。随着VZV在老年人和免疫受损个体中的细胞免疫功能下降(Gershon和Steinberg，1981；Burke等人，1982)，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)，经常合并慢性疼痛(带状疱疹后神经痛)、中风、失明和脊髓疾病。VZV在除潜伏病毒的神经细胞以外的所有人类细胞中复制。决定有效感染和潜伏感染的机制尚不清楚，但可能涉及病毒蛋白的特定细胞类型相互作用和宿主细胞抗病毒防御机制。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-NBS)相互作用，相互作用的结果有助于产生感染而不是潜伏期(Everett和Cherbi-Alix，2007)；此外，与神经元等有丝分裂后细胞中的PML-NB稳定性相比，经历有丝分裂的细胞中PML-NBS的结构发生了显著变化(Dellaire等人，2006a)。基于这些观察，我们假设VZV感染的结果(裂解与潜伏和随后的重新激活)是VZV蛋白与宿主细胞的抗病毒PML-NBS的细胞类型特异性相互作用的结果。为了验证这一假设，我们将：(1)利用免疫化学和共聚焦显微镜，分析感染VZV的非神经细胞是否存在PML-NBS与VZV基因61蛋白(VZV 61p)的相互作用，VZV基因61蛋白(VZV 61p)与已知与PML-NBS相互作用的疱疹病毒蛋白，以及VZV基因63蛋白(VZV 63p)，VZV基因63蛋白是在潜伏感染的人类神经节中表达的最普遍和丰富的VZV蛋白。检测PML、VZV 61p和VZV 63p的高表达和低表达对VZV基因表达和病毒滴度的影响：(目的2)利用Aim 1中的技术和荧光原位杂交技术检测神经节细胞中VZV DNA，分析PML-NBS与VZV 61p和VZV 63p之间的相互作用；和(AIM 3)使用AIM 2的技术分析在外植体诱导重新激活PML-NBS与VZV61p和VZV 63p相互作用的早期尸检中获得的人类神经节神经元。在获奖期间，Nagel博士将晋升为神经内科助理教授，这为内科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来加强培训。她将把80%的精力投入到研究中，20%投入到病人护理/临床教学中。她将在第四年的培训期间申请独立的R01奖项。她的主要科学导师将是科罗拉多丹佛大学神经学研究教授兰德尔·科尔斯博士，他是基础研究科学家，也是VZV发病机制的世界专家之一。尽管开发了带状疱疹疫苗，但即使每个60岁以上的美国人都接种了带状疱疹疫苗，每年仍将有超过50万新的带状疱疹病例发生。因此，了解导致VZV潜伏和重新激活的病毒和宿主细胞的相互作用将为预防VZV引起的严重神经系统疾病提供基础。

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.