The analysis of mechanism of Theiler's virus-induced damyelination in L* protein transgenic mice

泰勒氏小鼠脑脊髓炎病毒(TMEV)根据其生物学活性的不同分为两个亚型。DA菌株可导致易感品系小鼠脊髓慢性炎症性脱髓鞘。这是一种多发性硬化症的实验模型，人类中枢神经系统(CNS)中的脱髓鞘疾病。DA株持续感染和脱髓鞘的确切机制尚不清楚。DA株翻译17 kDa的蛋白质，命名为L*，与病毒的框外。为了阐明猪传染性支气管炎病毒持续感染和脱髓鞘的机制，本研究试图建立在不同启动子作用下表达L蛋白的转基因小鼠。我们构建了含有L蛋白基因的真核表达载体，它们分别位于MHC I类基因启动子、诱导型一氧化氮合酶基因和鸡β-肌动蛋白启动子下。将这些载体注射到FVB/NJ小鼠胚胎中。虽然在体外观察到了L*的强劲表达，但在体内并未证实L*的表达。因此，我们利用慢病毒载体获得了表达L蛋白的永生化巨噬细胞系J774。在疾病的慢性期，巨噬细胞被认为是TMEV的储存库。当J774感染DAL*-1时，DAL*-1不能合成L*，并减弱了中枢神经系统的脱髓鞘活性，病毒无法生长。相反，DAL*-1病毒能够在J774中生长，表达L*，这表明L*在巨噬细胞中的生长是重要的。此外，还构建了表达L*表位的突变病毒，并分析了L*在疾病急性期中枢神经系统的表达。在急性期，L*在易感和耐药小鼠的中枢神经系统均有表达，提示L*的表达本身不是慢性感染和脱髓鞘的决定因素。

Theiler's murine encephalomyelitis virus (TMEV) is classified into two subgroups based on the difference in biological activities. DA strain causes chronic inflammatory demyelination in the spinal cord in susceptible strains of mice. This serves as an experimental model of multiple sclerosis, human demyelinating disease in the central nervous system (CNS). The precise mechanism of persistent infection and demyelination by DA strain is yet to be elucidated. DA strain translates 17 kDa protein, designated L*, which is out of frame with the virus. To elucidate the mechanism of persistent infection of TMEV and demyelination, in this study we tried to generate transgenic mice expressing L* protein under different promoters. We generated the constructs which have L* protein cDNA under MHC class I or iNOS or chicken β-actin promoter. These constructs were injected into FVB/NJ mouse embryo. The expression of L* was not verified in vivo although robust expression of L* was observed in vitro. Therefore, we generated the immortalized macrophage cell line J774 expressing L* protein by using lentivirus vector. Macrophage is considered as the reservoir of TMEV in chronic phase of the disease. When J774 was infected with DAL*-1, which fails to synthesize L* and has attenuated demyelinating activity in the CNS, the virus failed to grow. In contrast, DAL*-1 virus was able to grow in J774 expressing L* indicating that L* is important to grow in macrophage. In addition, the mutant virus expressing epitope-tagged L* was generated and the L* expression in the CNS in acute phase of the disease was analyzed. In acute phase, L* was expressed in the CNS in both susceptible and resistant strains of mice suggesting that L* expression itself is not a determining factor of chronic infection and demyelination.