The Reoviridae include non-enveloped spherical virus particles, 700-1100 Angstroms in diameter, generally formed by concentric protein shells having T=13/icosahedral symmetry, which encapsidate 10-12 (1-4 kb) segments of a dsRNA genome. Our structural studies are focussed on two members of the family of Reoviridae, rotavirus and reovirus. Rotavirus infection results in severe infantile gastroenteritis and is the major cause of human infant mortality in developing countries. Although not a human pathogen, the closely related reoviruses have served as an important model system for studying the pathogenesis of viral infectious diseases. Infection by rotavirus and reovirus requires attachment of the surface hemagglutinin proteins (VP4 in rotavirus and sigma1 in reovirus) to surface receptors on cells lining the gut. For rotavirus, infection is mediated by trypsin cleavage of VP4, generating VP5* and VP8*. For reovirus, cell and tissue tropism are conferred by the sigma1 protein which is different for serotypes 1 (Lang) and 3 (Dearing). We recently used cryo-electron microscopy and icosahedral image reconstruction to derive the 3-dimensional structure of rhesus rotavirus under conditions which preserve the native conformational state of the protein and nucleic acid. The rich detail in the density maps demonstrates the power of this technique to reveal the architecture of complex macromolecular structures. Our objectives are to determine the supramolecular structure and design of several different rotavirus and reovirus particles as well as purify and crystallize VP4: 1. Rotavirus structure analysis a. Native versus spikeless (i.e., lacking the hemagglutinin VP4) rotavirus b. Compare strain SA11 and rhesus rotavirus to examine differences in VP4 c. Compare the structures of native and trypsin-cleaved rotavirus d. Examine crystalline tubes and sheets of the inner capsid protein, VP6 e. Perform immunolabeling using Fab fragments directed against the trypsin cleavage products of VP4, VP5* and VP8* f. Examine full (i.e., containing the dsRNA genome) and empty rotavirus cores 2. Reovirus structure analysis a. Compare the structures of native reovirus, trypsin-cleaved reovirus (intermediate subviral particles [ISVPs]) and reovirus cores b. Compare the structures of serotypes 1 (Lang) and 3 (Dearing) reovirus 3. Purify and crystallize VP4, the rotavirus hemagglutinin The structural information provided by our analyses will be fundamental for a complete understanding of mechanisms of viral pathogenesis and may provide important clues for the rational design of therapeutic strategies.
Showing the most recent 10 out of 14 publications
