Molecular Basis of TMEV Persistence

说明（申请人提供） 用Theiler鼠脑脊髓炎病毒（TMEV）对小鼠进行脑内接种（ic）提供了一种实验动物模型系统，其可用于理解持续CNS病毒感染如何导致慢性脱髓鞘，如多发性硬化（MS）中可能的情况。该研究的总体目标是阐明TMEV诱导的脱髓鞘疾病中病毒-宿主和病毒-细胞相互作用的分子基础。TMEV的持久性是“驱动”脱髓鞘过程所必需的，但脱髓鞘究竟是如何发生的仍然存在争议。我们认为，在脱髓鞘中，由MHC II类限制性CD 4 + Th 1 T细胞介导的病毒特异性DTH具有核心作用。被募集到中枢神经系统（CNS）中的单核细胞分化成巨噬细胞，巨噬细胞：（a）允许病毒持续存在和（B）导致髓鞘膜的旁观者损伤。然而，一旦TMEV建立了持续感染，病毒就会扩散到少突胶质细胞并有效感染，可能还有其他细胞。 最近，我们已经表明，TMEV复制是限制在巨噬细胞，但生产少突胶质细胞。 由于与巨噬细胞结合和/或感染巨噬细胞，TMEV诱导程序性细胞死亡（细胞凋亡），其特征在CNS白色物质中显著观察到。因此，病毒-巨噬细胞相互作用是TMEV持续存在的重要因素。 由于脱髓鞘需要TMEV持久性，我们想知道负责的病毒遗传元件，因为这可能提供持久性机制的见解。使用重组TMEV映射病毒持久性决定簇的编码衣壳的序列，和精细尺度映射表明，这个决定簇是构象的性质。因此，病毒-细胞受体相互作用也是TMEV持续存在的重要因素。在本项目中，我们计划鉴定GDVII病毒用于与细胞结合的附着因子，并进一步表征硫酸乙酰肝素蛋白聚糖在GDVII病毒感染中的作用（目的1），使用免疫学、分子和生物化学方法鉴定多年来一直抵抗鉴定的TMEV细胞受体（目的2），并进一步表征TMEV诱导的程序性细胞死亡（在鼠巨噬细胞中）（目的3）。

Description (provided by applicant) Intracerebral inoculation (ic) of mice with Theiler's murine encephalomyelitis virus (TMEV) provides an experimental animal model system that is useful in understanding how a persistent CNS virus infection leads to chronic demyelination, as might be the case in multiple sclerosis (MS). The overall goal of the proposed research is to elucidate the molecular basis of virus-host and virus-cell interactions in TMEV induced demyelinating disease. TMEV persistence is required to "drive" the demyelinating process, but exactly how demyelination occurs is still disputed. We believe that there is a central role for virusspecific DTH mediated by MHC class-II restricted CD4+ Th1 T cells in demyelination. Monocytes that are recruited into the central nervous system (CNS) differentiate into macrophages that: (a) allow viral persistence and (b) lead to bystander damage of myelin membranes. However, once TMEV establishes a persistent infection, the virus spreads to and productively infects oligodendrocytes, and possibly other cells. Recently, we have shown that TMEV replication is restricted in macrophages but productive in oligodendrocytes. As a result of binding to and/or infection of macrophages, TMEV induces programmed cell death (apoptosis), the hallmarks of which are prominently observed in the CNS white matter. Thus, the virus-macrophage interaction is an important element in TMEV persistence. Since TMEV persistence is required for demyelination, we wanted to know the responsible viral genetic elements as this might provide insight into the mechanism(s) of persistence. Use of recombinant TMEV mapped a viral persistence determinant to sequences encoding the capsid, and fine scale mapping suggested that this determinant is conformational in nature. Thus, the virus-cell receptor interaction is also an important element in TMEV persistence. In this Project, we plan to identify the attachment factor GDVII virus uses to bind to cells and further characterize the role of heparan sulfate proteoglycans in GDVII virus infection (aim 1), use immunological, molecular and biochemical approaches to identify the TMEV cellular receptor that has resisted identification over the years (aim 2), and further characterize TMEV-induced programmed cell death ( in murine macrophages (aim 3).