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.
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