This fellowship application proposes studies to understand the pathogenic mechanism by which rotavirus causes severe diarrhea in children. Rotavirnses have a unique morphogenesis in which particles obtain a transient membrane envelope that is formed by the budding of newly made subviral particles into the endoplasmic reticulum (ER). This process is mediated by a viral nonstructural glycoprotein called NSP4, which is localized to the ER membrane of infected cells. Recent studies in this laboratory have shown that NSP4 and a synthetic peptide NSP4 114.135 (corresponding to NSP4 amino acids 114-135), when injected intraperitoneally or intraduodenally, induced diarrhea in young mice and rats. NSP4 and NSP4 114.1350, when expressed in or added exogenously to insect cells, mobilize intracellular calcium. We speculate that NSP4 may be the virulence factor responsible for inducing the diarrhea caused by rotaviruses, and that the calcium mobilization property of NSP4 may underlie a signal transduction pathway which contributes to virus pathogenicity. Electrophysiological approaches as well as conventional molecular biological techniques are proposed in this application to delineate the mechanisms by which NSP4 mobilizes intracellular calcium and induces diarrhea in young animals. Understanding these mechanisms may provide the key to resolving the pathogenic pathway of rotaviruses. Because of the unique properties of this rotavirus nonstructural glycoprotein, we are likely to elucidate new mechanisms of viral pathogenesis and novel signal transduction pathways of calcium mobilization caused by viral proteins. In the proposed research, I will learn new techniques including: Ussing chamber, planar lipid bilayer, construction and use of recombinant plasmids or virus, point specific mutations, and other molecular approaches necessary to carry out research to answer fundamental questions in molecular biology.
