GLYCOPROTEIN FUNCTIONS IN PATHOGENESIS OF CALIFORNIA ENCEPHALITIS

拉克罗斯病毒(LAC)，是美国 布尼亚病毒属(布尼亚病毒科)是一种公认的人类病原体 对美国中西部的许多儿童脑炎病例负有责任， 是研究本亚病毒致病机制的重要模型。我们的 实验室研究LAC病毒神经发病机制已有数年之久， 尤其是病毒毒力的遗传决定因素(由 Griot等人，1993a)。这些研究勾勒出了顺序步骤 参与中枢神经系统疾病的发展(Janssen等人，1984)和 展示了中间(M)RNA片段在病毒传播中的关键作用 和毒力(Janssen等人，1986 Griot等人，1993b)。 我们建议将我们的研究扩展到几个方面，这些研究将 补充了我们的基因发现。在第一个具体目标中，我们将使用 杆状病毒表达的重组糖蛋白进一步确定 病毒结合、进入和神经侵袭之间的关系。一个 可溶形式的G1，LAC病毒附着蛋白已被用作 病毒蛋白的替代品，因为它寡聚，结合易感 细胞，并抑制病毒感染。该系统将用于地图绘制 参与受体结合的G1结构域，并检验这一假说 G1-受体相互作用的差异在 确定致病性。在第二个具体目标中，我们将确定 负责融合的结构域，我们认为这是由G 蛋白。为了实现这一目标，我们将引入截断，并 最终定位突变为LAC M ORF，表达于 牛痘疫苗系统。这些构造将用于标识关键的 负责融合的区域使用定量分析，并进行比较 神经侵袭性LAC病毒与非侵袭性LAC病毒的融合功能 侵略型塔希纳。 在第三个具体目标中，我们将使现有模型适用于其他 包括布尼亚病毒在内的负链病毒(Whelan等人，1995； Lawson等人，1995年；Bridgen和Elliot，1996)，以开发一种用于 重组病毒的产生。这将允许我们将 来自前两个目标的发现涉及到复制病毒粒子的背景， 并确认病毒基因组的特定区域在其 致病性。总之，这些实验将加深我们对 特异糖蛋白结构域和功能在细胞周期中的作用 神经侵袭性和神经毒性。

La Crosse virus (LAC), a member of the California serogroup of the bunyavirus genus (Family Bunyaviridae), is an established human pathogen responsible for many cases of pediatric encephalitis in the midwestern US, and it is an important model for the study of bunyavirus pathogenesis. Our laboratory has studied LAC virus neuropathogenesis for several years, and particularly the genetic determinants of viral virulence (Reviewed by Griot et al., 1993a). These studies have delineated the sequential steps involved in the development of CNS disease (Janssen et al., 1984) and demonstrated the key roles of the middle (M) RNA segment in virus spread and virulence (Janssen et al., 1986 Griot et al., 1993b). We propose to extend our studies with several lines of research that will complement our genetic findings. In the first specific aim, we will use baculovirus-expressed recombinant glycoproteins to further define the relationship between viral binding, entry, and neuroinvasiveness. A soluble form of G1, the LAC viral attachment protein has been used as a surrogate for the viral protein as it oligomerizes, binds susceptible cells, and inhibits viral infection. This system will be used map the domains of G1 involved in receptor binding, and to test the hypothesis that differences in the G1-receptor interaction are important in determining pathogenicity. In the second specific aim, we will identify the domains responsible for fusion, which we believe is mediated by the G protein. To perform this aim, we will introduce truncations, and eventually site directed mutations into the LAC M ORF expressed in a vaccinia system. These constructs will be used to identify the critical regions responsible for fusion using quantitative assays, and to compare the fusion function of the neuroinvasive LAC virus with that of the non- invasive Tahyna. In the third specific aim, we will adapt existing models for other negative stranded viruses including bunyaviruses (Whelan et al., 1995; Lawson et al., 1995; Bridgen and Elliot, 1996) to develop a system for the generation of recombinant viruses. This will allow us to place the findings from the first two aims into the context of a replicating virion, and to confirm the role of specific regions of the viral genome in its pathogenicity. Together these experiments will further our understanding the role of specific glycoprotein domains and functions in neuroinvasiveness and neurovirulence.