Theiler's virus-induced aoptosis: A mechanism for CNS virus persistence

描述（由申请人提供）：Theiler的小鼠脑脊髓炎病毒（TMEV）是一种高度溶细胞的RNA病毒，可在易感小鼠株中产生持续的中枢神经系统（CNS）感染和免疫介导的脱髓鞘。TMEV感染为多发性硬化症提供了相关的实验动物模型。与宿主细胞存活的非溶细胞RNA病毒的持久性不同，溶细胞RNA病毒的持久性（被感染细胞死亡）需要持续的细胞间传播以维持感染。在这种情况下，理想情况下，病毒持续存在的靶细胞应该是可再生的。感染不应是高度多产的；然而，无论如何，病毒特异性宿主免疫反应可能控制更多产的感染。巨噬细胞是主要的病毒库，并提供了TMEV在小鼠中枢神经系统中持续存在的体外模型。感染TMEV的巨噬细胞发生凋亡并限制病毒产生（<10 pfu/细胞），而感染其他啮齿动物细胞，包括神经元和少突胶质细胞，坏死细胞死亡与高病毒产量（200-500 pfu/细胞）相关。TMEV感染是诱导小鼠巨噬细胞凋亡的必要条件，因为UV灭活病毒吸附在细胞表面引发感染并不诱导细胞凋亡。我们已经证明，TMEV感染通过Bax介导的内在途径诱导小鼠巨噬细胞凋亡，并严重限制感染性病毒的产生。我们的工作假设是，tmev诱导的细胞凋亡是一种宿主细胞特异性机制，提供了一种将病毒传播到未感染的巨噬细胞的手段，这些巨噬细胞吞噬含有感染性病毒的凋亡残留物。为了了解TMEV感染如何触发小鼠巨噬细胞凋亡并限制感染性病毒的产生，我们提出了以下四个具体目标：1)完成TMEV病毒感染M1-D巨噬细胞内在凋亡通路上游信号的鉴定，确定耐药株和易感株小鼠原代巨噬细胞凋亡的潜在差异；2)抑制感染小鼠的细胞凋亡，评估其对TMEV CNS持久性、病毒特异性CD4+ T细胞应答和脱髓鞘疾病的影响，并检测p53和Noxa缺失转基因小鼠对CNS持久性和脱髓鞘疾病的影响；3)通过在哺乳动物细胞中表达TMEV非结构蛋白L和3CD，鉴定病毒潜在的细胞凋亡“触发器”，并确定衣壳蛋白缺失的TMEV复制子是否能诱导细胞凋亡；4)研究小鼠巨噬细胞中感染性病毒丢失与细胞凋亡发生的相关机制。

DESCRIPTION (provided by applicant): Theiler's murine encephalomyelitis virus (TMEV) is a highly cytolytic RNA virus that produces persistent central nervous system (CNS) infection and immune-mediated demyelination in susceptible strains of mice. TMEV infection provides a relevant experimental animal model for multiple sclerosis. In contrast to persistence of non-cytolytic RNA viruses in which the host cell survives, persistence of lytic RNA viruses in which an infected cell dies requires continuous cell- to-cell spread in order to maintain the infection. In this circumstance, the target cell in which the virus persists ideally should be renewable. The infection should not be highly productive; however, virus-specific host immune responses may control a more productive infection anyway. Macrophages are the principal virus reservoir and provide an in vitro model for TMEV persistence in the mouse CNS. TMEV infected macrophages undergo apoptosis and restrict virus production (<10 pfu/cell), in contrast to infection in other rodent cells, including neurons and oligodendrocytes, where necrotic cell death is associated with high virus yields (200-500 pfu/cell). TMEV infection is required to induce apoptosis in murine macrophages since UV- inactivated virus adsorbed to the cell surface to initiate infection does not induce apoptosis. We have shown that TMEV infection induces apoptosis through the intrinsic pathway that is Bax- mediated and severely restricts infectious virus production in murine macrophages. Our working hypothesis is that TMEV-induced apoptosis is a host cell-specific mechanism providing a means of spreading virus to uninfected macrophages that phagocytose apoptotic remnants containing infectious virus. To understand how TMEV infection triggers apoptosis in murine macrophages and restricts infectious virus production, four specific aims are proposed: 1) finish the identification of upstream signals (apical to the mitochondrion) involved in the intrinsic apoptotic pathway during TMEV virus infection in M1-D macrophages and determine potential differences in apoptosis in primary macrophages from resistant and susceptible strains of mice; 2) inhibit apoptosis in infected mice to assess the effect on TMEV CNS persistence, virus-specific CD4+ T cell responses and demyelinating disease, and examine the effect in transgenic mice deficient in p53 and Noxa on CNS persistence and demyelinating disease; 3) identify potential virus "triggers" of apoptosis by expressing TMEV nonstructural proteins L and 3CD in mammalian cells, and determine whether TMEV replicons in which the capsid proteins are deleted can induce apoptosis; and 4) investigate the mechanism of loss of infectious virus in murine macrophages that is associated with onset of apoptosis. PUBLIC HEALTH RELEVANCE: The notion that multiple sclerosis (MS) is an autoimmune disease is widely accepted as fact. While MS is undoubtedly immune-mediated, an autoimmune mechanism remains unproven. Circumstantial evidence points to a persistent virus infection in MS. Moreover, immune- mediated damage can result from a virus infection in which the host immune response is directed to virus proteins rather than self proteins (autoimmunity). Theiler's murine encephalomyelitis virus (TMEV) produces a persistent central nervous system infection and immune-mediated demyelination in susceptible strains of mice, providing a relevant experimental animal model for MS. TMEV persistence is needed to drive the demyelinating disease process, but just how this virus that rapidly kills infected cells is able to perpetuate the infection is not known. This proposal addresses a mechanism of TMEV persistence, programmed cell death also termed apoptosis, which decreases virus production (may be required for persistence of virulent virus) and also fosters cell-to-cell virus spread (required for persistence in the presence of host immunity).