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

Journal Article · · Virology Journal
 [1];  [2];  [3];  [4];  [5]
  1. Texas A & M Univ., College Station, TX (United States). Dept. of Pathobiology; DOE/OSTI
  2. Texas A & M Univ., College Station, TX (United States). Dept. of Pathobiology; Texas State Veterinary Diagnostic Lab., College Station, TX (United States)
  3. Texas A & M Univ., College Station, TX (United States). Dept. of Pathobiology; Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
  4. Texas A & M Univ., College Station, TX (United States). Dept. of Physiology and Pharmacology
  5. Texas A & M Univ., College Station, TX (United States). Dept. of Pathobiology; Stephen F. Austin State Univ., Nacogdoches, TX (United States). Dept. of Biology
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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 (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. Results: Mutations 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. 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.
Research Organization:
Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)
Sponsoring Organization:
National Institutes of Health (NIH); USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
Grant/Contract Number:
NA0003525
OSTI ID:
1626613
Journal Information:
Virology Journal, Journal Name: Virology Journal Journal Issue: 1 Vol. 10; ISSN 1743-422X
Publisher:
BioMed CentralCopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (3)

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