Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1.
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 (NSP446-175-ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP4(46-175)-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.


RESULTS
Mutations of the hydrophilic face (NSP4(46-175)-Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP4(46-175)-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.


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.
Department of Pathobiology, Texas A&M University, TVMC, College Station, Texas 77843-4467, USA. rebeccadparr@gmail.com.
Judith M
JM
Ball
Megan E
ME
Schroeder
Cecelia V
CV
Williams
Friedhelm
F
Schroeder
Rebecca D
RD
Parr
D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites; D051242:Caveolin 1 / Q000378:metabolism*; D004252:DNA Mutational Analysis; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D014118:Toxins, Biological / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D051242:Caveolin 1; D006023:Glycoproteins; D050505:Mutant Proteins; C081369:NS28 protein, rotavirus; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins