Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1

Abstract

Background: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction. Methods. A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed to alanine (NSP4§ssub§46- 175§esub§-ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP4§ssub§46-175§esub§- HydroMut). In total, twelve mutants of NSP4 were generated to define the cav-1 binding site. Synthetic peptides corresponding to the hydrophobic and charged faces of NSP4 were examined for structural changes by circular dichroism (CD) and diarrhea induction by a neonatal mouse study. Results: Mutations of the hydrophilic face (NSP4§ssub§46-175§esub§-Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP4§ssub§46-175§esub§-HydroMut) failed to bind cav-1. These data suggest NSP4 and cav-1 associate via a hydrophobic interaction. Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue. Both NSP4§ssub§HydroMut112-140,§esub§ that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4§ssub§AlaAcidic112-140§esub§ that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea. Whereas peptides NSP4wild type §ssub§112§esub§ §ssub§-140§esub§ and NSP4§ssub§AlaBasic112-140§esub§ that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea. Conclusions: These data show that the cav-1 binding domain is within the hydrophobic face of the NSP4 amphipathic helix. The integrity of the helical structure is important for both cav-1 binding and diarrhea induction implying a connection between NSP4 functional and binding activities. © 2013 Ball et al.; licensee BioMed Central Ltd.