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

Judith M Ball,Rebecca D Parr,Friedhelm Schroeder,Megan E Schroeder,Cecelia V Williams

doi:10.1186/1743-422x-10-336

Abstract

BackgroundRotavirus (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.MethodsA 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 (NSP446-175-ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP446-175-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.ResultsMutations of the hydrophilic face (NSP446-175-Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP446-175-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 NSP4HydroMut112-140, that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea. Whereas peptides NSP4wild type 112−140 and NSP4AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea.ConclusionsThese 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.

Highlights

Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents
The hydrophobic face of the NSP4 amphipathic alpha helix (AAH) binds cav-1 To identify the face of the amphipathic helix that binds cav-1, a 3-D structural model was generated using the crystallographic determinants for NSP4 95-135 [42] and visualized using PyMol (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC)
The six charged residues between 114 and 135 were mutated to alanine (D114A, K115A, R119A, E125A, D132A, and K133A) in FLNSP4-Ala6, and three hydrophobic residues were mutated to charged residues (I113R, V124K, and Y131D), in FLNSP4-HydroMut (Figure 1A and B, Table 1)

Summary

Introduction

Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. RV NSP4 is the first described viral enterotoxin and induces early secretory diarrhea in rodents [1,3,4,5,6]. Reports show NSP4 is an ER transmembrane glycoprotein that serves as an intracellular receptor that binds double layered particles [13,14,15,16], and facilitates entry into the ER and acquisition of the outer coat to form triple layered particles with a transient ER membrane [10,15,16,17]. NSP4 appears to function in viral pathogenesis, replication, and morphogenesis, as well as serving as an enterotoxin that induces calcium signaling events and fluid loss [20]

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