Membrane interactions of a novel viral enterotoxin: Rotavirus nonstructural glycoprotein NSP4

The rotavirus enterotoxin, NSP4, is a novel secretory agonist that also pla ys a role in the unique rotavirus morphogenesis that involves a transient b udding of newly made immature viral particles into the endoplasmic reticulu m, NSP4 and an active peptide corresponding to NSP4 residues 114 to 135 (NS P4(114-135)) mobilize intracellular calcium and induce secretory chloride c urrents when added exogenously to intestinal cells or mucosa. Membrane-NSP4 interactions may contribute to these alterations; however, details of a li pid-binding domain are unresolved. Therefore, circular dichroism was used t o determine (i) the interaction(s) of NSP4 and NSP4114-135 With model membr anes, (ii) the conformational changes elicited in NSP4 upon interacting wit h membranes, (iii) if NSP4(114-135) is a membrane interacting domain, and ( iv) the molar dissociation constant (K-d) Of NSP4(114-135) With defined lip id vesicles. Circular dichroism revealed for the first time that NSP4 and N SP4(114-135) undergo secondary structural changes upon interaction with mem brane vesicles. This interaction was highly dependent on both the membrane surface curvature and the lipid composition. NSP4 and NSP4(114-135) prefere ntially interacted with highly curved, small unilamellar vesicle membranes (SUV), but significantly less with low-curvature, large unilamellar vesicle membranes (LUV), Binding to SUV, but not LUV, was greatly enhanced by nega tively charged phospholipids. Increasing the SW cholesterol content, concom itant with the presence of negatively charged phospholipids, further potent iated the interaction of NSP4(114-135) With the SUV membrane. The K-d of NS P4(114-135) was determined as well as partitioning of NSP4(114-135) With SU Vs in a filtration-binding assay. These data confirmed NSP4 and its active peptide interact with model membranes that mimic caveolae.