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

Ball JM, Schroeder ME, Williams CV, Schroeder F, Parr RD - Virol. J. (2013)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, Texas A&M University, TVMC, College Station, Texas 77843-4467, USA. rebeccadparr@gmail.com.

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 (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.

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Rotavirus NSP4 protein alignments. NSP4 protein sequences obtained from GenBank of twelve rotavirus strains that represent groups A through E. [GenBank: ABZ04174, BAA24144, ABZ04170, BAB83830, AAA64924, AAL11029, AAB58698, AAD50676, ABV66094, BAA13728, P08434, AND P04512] were aligned with CLUSTALW for multiple sequence alignments [51,52].
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Figure 6: Rotavirus NSP4 protein alignments. NSP4 protein sequences obtained from GenBank of twelve rotavirus strains that represent groups A through E. [GenBank: ABZ04174, BAA24144, ABZ04170, BAB83830, AAA64924, AAL11029, AAB58698, AAD50676, ABV66094, BAA13728, P08434, AND P04512] were aligned with CLUSTALW for multiple sequence alignments [51,52].

Mentions: NSP4 amino acids 113-135 obtained from GenBank of twelve RV strains that represent groups A through E. (accession no. ABZ04174, BAA24144, ABZ04170, BAB83830, AAA64924, AAL11029, AAB58698, AAD50676, ABV66094, BAA13728, P08434, AND P04512) were aligned with CLUSTAL (FigureĀ 6). The following observations were made: (i) all aligned sequences included I113 and L127 except Human RV C I113M and L127K; (ii) all twelve strains contained L116; (iii) position 124 was V with one exception, Human RV C V124D; (iv) L134 was conserved with the exception of EDIM L134M and Human RV C L134I, another hydrophobic amino acid; and the residue 131 varied from Y131, H131 (NCDV, OSU, Wa, RRV), S131 (Human RV C). The high similarity of I113, L116, V124, L127, and L134 infer the importance of these residues. Given the high sequence divergence recently reported in NSP4 sequences, the conservation of the hydrophobic residues is all the more remarkable [44].


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

Ball JM, Schroeder ME, Williams CV, Schroeder F, Parr RD - Virol. J. (2013)

Rotavirus NSP4 protein alignments. NSP4 protein sequences obtained from GenBank of twelve rotavirus strains that represent groups A through E. [GenBank: ABZ04174, BAA24144, ABZ04170, BAB83830, AAA64924, AAL11029, AAB58698, AAD50676, ABV66094, BAA13728, P08434, AND P04512] were aligned with CLUSTALW for multiple sequence alignments [51,52].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924327&req=5

Figure 6: Rotavirus NSP4 protein alignments. NSP4 protein sequences obtained from GenBank of twelve rotavirus strains that represent groups A through E. [GenBank: ABZ04174, BAA24144, ABZ04170, BAB83830, AAA64924, AAL11029, AAB58698, AAD50676, ABV66094, BAA13728, P08434, AND P04512] were aligned with CLUSTALW for multiple sequence alignments [51,52].
Mentions: NSP4 amino acids 113-135 obtained from GenBank of twelve RV strains that represent groups A through E. (accession no. ABZ04174, BAA24144, ABZ04170, BAB83830, AAA64924, AAL11029, AAB58698, AAD50676, ABV66094, BAA13728, P08434, AND P04512) were aligned with CLUSTAL (FigureĀ 6). The following observations were made: (i) all aligned sequences included I113 and L127 except Human RV C I113M and L127K; (ii) all twelve strains contained L116; (iii) position 124 was V with one exception, Human RV C V124D; (iv) L134 was conserved with the exception of EDIM L134M and Human RV C L134I, another hydrophobic amino acid; and the residue 131 varied from Y131, H131 (NCDV, OSU, Wa, RRV), S131 (Human RV C). The high similarity of I113, L116, V124, L127, and L134 infer the importance of these residues. Given the high sequence divergence recently reported in NSP4 sequences, the conservation of the hydrophobic residues is all the more remarkable [44].

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, Texas A&M University, TVMC, College Station, Texas 77843-4467, USA. rebeccadparr@gmail.com.

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 (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.

Show MeSH
Related in: MedlinePlus