GLYCOPROTEIN FUNCTIONS IN PATHOGENESIS OF CALIFORNIA ENCEPHALITIS

拉克罗斯病毒（LAC），一个成员的加州血清组的 布尼亚病毒属（布尼亚病毒科），是一种公认的人类病原体 导致了美国中西部的许多儿童脑炎病例， 是研究布尼亚病毒致病机理的重要模型。我们 实验室已经研究了LAC病毒的神经发病机制数年， 特别是病毒毒力的遗传决定因素（由 Griot等人，1993年a）。这些研究描绘了 参与CNS疾病的发展（Janssen等，（1984年） 证明了中间（M）RNA片段在病毒传播中的关键作用 和毒力（Janssen等人，1986年Griot等人，1993年b）。 我们建议扩展我们的研究与几个线的研究， 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，以进一步定义 病毒结合、进入和神经侵袭性之间的关系。一 作为G1的可溶形式，LAC病毒附着蛋白已被用作 病毒蛋白的替代物，因为它寡聚化， 细胞，并抑制病毒感染。该系统将用于绘制 参与受体结合的G1结构域，并检验这一假设 G1-受体相互作用的差异对于 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， 最终定点突变到表达在一个细胞中的LAC M ORF中， 牛痘系统这些结构将用于确定关键的 负责融合的区域，并比较 神经侵袭性LAC病毒与非神经侵袭性LAC病毒的融合功能 入侵的塔海纳 在第三个具体目标中，我们将调整现有模型， 负链病毒包括布尼亚病毒（Whelan等，一九九五年; 劳森等人，1995年; Bridgen和Elliot，1996年）开发了一个系统， 重组病毒的产生。这将使我们能够将 将前两个目标的发现转化为复制病毒体的背景， 并确认病毒基因组的特定区域在其 致病性这些实验将进一步加深我们对 特异性糖蛋白结构域和功能在 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.