Neurovirulence of murine coronavirus: roles of viral genes

描述（由申请人提供）：鼠冠状病毒，小鼠肝炎病毒（MHV）引起小鼠急性脑炎，随后引起慢性脱髓鞘疾病;后者是研究人类脱髓鞘疾病（如多发性硬化症）的公认动物模型。由MHV诱导的神经毒力的程度取决于病毒和宿主因素;病毒株在毒力和嗜性方面不同，并且多种病毒基因有助于毒力的程度。我们已经使用嵌合重组病毒的基因来源于高度嗜神经性JHM株和较少嗜神经性A59株进行研究，在小鼠模型中，神经毒力的病毒决定因素。这些研究表明，刺突蛋白，病毒附着和传播的介质，显然是神经毒力的主要贡献者;然而，也很清楚，其他病毒基因在决定神经毒力的程度中发挥重要作用。我们在这里测试的假设，其他结构基因以及非结构基因有助于神经毒力。我们建议从MHV的JHM株构建感染性cDNA克隆;然后我们将使用该cDNA沿着现有的A59全长cDNA来回收具有确定交叉点的嵌合病毒。我们将使用这些病毒来确定神经毒力中结构基因与非结构基因的重要性。然后，我们将集中在两个非必需的蛋白质，血凝素酯酶（HE）和ORF 2a编码的30 kDa蛋白在神经毒力的作用，有初步的数据表明，这两种蛋白质确实影响毒力，可能通过不同的机制。高神经毒性JHM株表达HE蛋白，但A59株不表达。我们将继续正在进行的研究表明，表达HE确实增加了中枢神经系统的毒力，并开始解决这种增强的机制。ORF 2a中编码的30 kDa蛋白质对2组冠状病毒具有特异性;我们最近观察到该蛋白质中的一个氨基酸取代导致重组MHV-A59的减毒。我们将通过研究ORF 2a表达被消除或其基因产物突变的病毒的发病机制来追求这些发现。重点将是ORF 2a蛋白在增强神经毒力中的作用，可能是通过与宿主免疫反应相互作用。我们相信，这些研究将导致更好地了解MHV感染期间的神经毒力的决定因素。

DESCRIPTION (provided by applicant): The murine coronavirus, mouse hepatitis virus (MHV) causes acute encephalitis and, followed by a chronic demyelinating disease in mice; the latter is a recognized animal model for the study of human demyelinating diseases, such as multiple sclerosis. The extent of neurovirulence induced by MHV is dependent on both viral and host factors; viral strains differ in virulence and tropism and multiple viral genes contribute to the extent of virulence. We have used chimeric recombinant viruses with genes derived from the highly neurotropic JHM strain and the less neurotropic A59 strain to carry out studies, in the mouse model, of the viral determinants of neurovirulence. These studies have shown that the spike protein, the mediator of viral attachment and spread, is clearly a major contributor to neurovirulence; however, it is also clear that other viral genes play important roles in determining the extent of neurovirulence. We test here the hypothesis that other structural genes as well as non-structural genes contribute to neurovirulence. We propose to construct an infectious cDNA clone from the JHM strain of MHV; we will then use this cDNA along with an existing A59 full length cDNA to recover chimeric viruses with defined crossover points. We will use these viruses to determine the importance of structural versus nonstructural genes in neurovirulence. We will then focus on the roles of two non essential proteins, hemagglutinin esterase (HE) and the ORF 2a encoded 30 kDa protein in neurovirulence; there are preliminary data indicating that both of these proteins do influence virulence, likely by different mechanisms. The HE protein is expressed by the highly neurovirulent JHM strain but not the A59 strain. We will continue ongoing studies indicating that expression of HE does indeed increase virulence in the central nervous system and begin to address the mechanism of this enhancement. The 30kDa protein encoded in ORF2a is specific to group 2 coronaviruses; we have recently observed that one amino acid substitution within this protein results in attenuation of recombinant MHV-A59. We will pursue these findings by investigating the pathogenesis of viruses in which expression of ORF2a has been eliminated or its gene product mutated. The focus will be on a role for the ORF2a protein in enhancing neurovirulence, possibly through interacting with the host immune response. We believe these studies will lead to a better understanding of the determinants of neurovirulence during MHV infection.