The rotavirus nonstructural protein 4 (NSP4) interacts with both the N- and C- termini of caveolin-1

Abstract

Rotavirus (RV) is an etiologic agent of viral gastroenteritis in children and infants worldwide, accounting for an estimated 500,000 deaths annually. NSP4, the first described viral enterotoxin, contributes to RV pathogenesis by mobilizing intracellular calcium through multiple mechanisms that promote abnormal ion transport and subsequent secretory diarrhea. NSP4 and the enterotoxic peptide 114-135 preferentially interact with model membranes mimicking caveolae in lipid composition and radius of curvature. Our laboratory has recently reported the colocalization and coimmunoprecipitation of NSP4 with caveolin-1, the structural protein of caveolae. Moreover, the caveolin-1 binding domain of NSP4 has been localized to the enterotoxic peptide. We now report that caveolin-1 binds NSP4 via the N- and C-termini and one terminus is sufficient for binding. A panel of caveolin-1 deletion mutants was expressed in a yeast two-hybrid assay against an NSP4 bait. Caveolin-1 mutants retaining at least one terminus were capable of binding the NSP4 bait. An in vitro binding assay confirmed the two-hybrid results and localized the NSP4 binding domains to caveolin-1 residues 2-22 and 161-178. These data support the hypothesis that caveolin-1 mediates NSP4 signaling and/or intracellular trafficking.