LaCrosse (LAC) virus is a member of the California serogroup of the Bunyaviridae. LAC is an enveloped virus, containing two viral glycoproteins (G1 and G2) external to a lipid membrane. Within this envelope, three single-stranded RNA genome segments (L, M and S) are complexed with the nucleocapsid protein, N and the viral polymerase, L. Preliminary experiments utilizing cryo- electron microscopy have resulted in images of LAC virions, cryo- preserved in amorphous ice, containing structural information not present in the images of negative stained particles. This cryo- technique allows one to prepare frozen, hydrated and unstained biological specimens in different chemical environments and to eliminate the potential artifacts generally encountered in conventional methods. Computer analysis of the digitized images has led to the tentative conclusion that the LAC virion possesses icosahedral symmetry. The present proposal is aimed at studying the structure-function relationships of the intact LAC virion and its macromolecular components by a combination of cryo-electron microscopy, computerized image reconstruction and biochemical analysis. The first part of the proposal is designed to produce a three-dimensional reconstruction of the LAC virion, including a description to the geometrical arrangement of the glycoprotein spikes at neutral and acidic pH. These structural data will provide information about the stable architecture of the virion as well as its potentially different conformations under conditions where cell fusion occurs at low pH. The second part of the proposal is to determine the 3-dimensional arrangement of the nucleocapsids within the intact virion. This information can be used to understand the structural stability of a virion which has no matrix protein and to deduce possible mechanisms for viral assembly. The third part of the proposal is to study the molecular interactions of N protein with viral RNA species. These data will enhance our understanding of the packaging of the viral genome and the potential role of N protein in the regulation of transcription and replication.
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