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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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Representative Western blot analyses of yeast lysates co-transformed with NSP4 or NSP4 mutants with caveolin-1 fusion proteins. The presence of NSP4- and cav-1 fusion proteins in the Y2H assay was shown by Western blot analyses of the yeast co-transformed with pD22cav-1 and each of the following fusion proteins: pD32NSP4 46-175 (lanes 2A and 2B), pD32NSP4-Ala6 (lanes 3A and 3B) , pD32NSP4-HydroMut (lane 4), pD32Rev113I (lane 5), pD32Rev124V (lane 6), and pD32Rev131Y (lane 7). Lanes 8 and 9 show the FLNSP4-HydroMut and FLNSP4, respectively. Blots were probed using (A) rabbit anti-NSP4150-175 or (B) rabbit anti-cav-12-31. Panel A: The non-transformed yeast (MaV203, lane 1) showed a non-specific band at ~29 kD that was observed in all lanes. Lysates expressing FLNSP4-HydroMut (lane 8) demonstrated NSP4-specific bands at ~ 28, 24 kD. Lysates expressing FLNSP4 showed bands at ~ 28, 24, and 20 kD (lane 9). All of the co-transformed yeast demonstrated a NSP4-fusion protein band at ~ 34.4 kD (A, lanes 2-7). Panel B revealed a faint contaminating band below the 34.9 kD specific caveolin-1-fusion protein band (lanes 1-7). Lanes 2-7 show the specific caveolin-1 fusion protein band at ~34.9 kD. Panel C: Lysates expressing FLNSP4 (lane 1) showed bands at ~ 24 and 28 kD and (lane 9). All of the co-transformed yeast demonstrated both monomeric and multimeric NSP4-specific fusion protein bands (lanes 1). Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed the ~34.9 kD specific NSP4-1-fusion protein band (lanes 2-5). Also, Rev1M124,127A demonstrated a multimeric NSP4-fusion specific band similar to one observed with FLNSP4. Panel D: Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed a ~ 34.9 kD specific caveolin-1-fusion protein band (lanes 1-4). Also, Rev1M124,127A demonstrated a multimeric caveolin-1-fusion protein band.
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Figure 3: Representative Western blot analyses of yeast lysates co-transformed with NSP4 or NSP4 mutants with caveolin-1 fusion proteins. The presence of NSP4- and cav-1 fusion proteins in the Y2H assay was shown by Western blot analyses of the yeast co-transformed with pD22cav-1 and each of the following fusion proteins: pD32NSP4 46-175 (lanes 2A and 2B), pD32NSP4-Ala6 (lanes 3A and 3B) , pD32NSP4-HydroMut (lane 4), pD32Rev113I (lane 5), pD32Rev124V (lane 6), and pD32Rev131Y (lane 7). Lanes 8 and 9 show the FLNSP4-HydroMut and FLNSP4, respectively. Blots were probed using (A) rabbit anti-NSP4150-175 or (B) rabbit anti-cav-12-31. Panel A: The non-transformed yeast (MaV203, lane 1) showed a non-specific band at ~29 kD that was observed in all lanes. Lysates expressing FLNSP4-HydroMut (lane 8) demonstrated NSP4-specific bands at ~ 28, 24 kD. Lysates expressing FLNSP4 showed bands at ~ 28, 24, and 20 kD (lane 9). All of the co-transformed yeast demonstrated a NSP4-fusion protein band at ~ 34.4 kD (A, lanes 2-7). Panel B revealed a faint contaminating band below the 34.9 kD specific caveolin-1-fusion protein band (lanes 1-7). Lanes 2-7 show the specific caveolin-1 fusion protein band at ~34.9 kD. Panel C: Lysates expressing FLNSP4 (lane 1) showed bands at ~ 24 and 28 kD and (lane 9). All of the co-transformed yeast demonstrated both monomeric and multimeric NSP4-specific fusion protein bands (lanes 1). Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed the ~34.9 kD specific NSP4-1-fusion protein band (lanes 2-5). Also, Rev1M124,127A demonstrated a multimeric NSP4-fusion specific band similar to one observed with FLNSP4. Panel D: Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed a ~ 34.9 kD specific caveolin-1-fusion protein band (lanes 1-4). Also, Rev1M124,127A demonstrated a multimeric caveolin-1-fusion protein band.

Mentions: To verify the presence of the NSP4 and cav-1 GAL-4 fusion proteins in the yeast that showed no binding to cav-1 in the Y2H assay, the co-transformed lysates were probed with either anti-NSP4150-175 (Figure 3A and C) or anti-cav-12-31 (Figure 3B and D) peptide-specific antibodies. Controls included untransformed MaV203 yeast lysates. The controls failed to show an antibody-specific band at the correct molecular weight of the fusion proteins for both the activating domain (AD)-NSP4 and binding domain (BD)-cav-1. However, a non-specific band at ~28 kD was observed when blotted with both antisera (Figure 3, lane 1). All NSP4 and cav-1 fusion proteins tested expressed at the correct molecular weight (34.4 kD and 34.9 kD, respectively) and were reactive with NSP4- (Figure 3, panels A&C) and cav-1 peptide-specific antisera (panel B&D). These data established that the encoded sequences from both plasmids were translated as fusion proteins in the co-transformed yeast, even when no reactivity was detected.


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)

Representative Western blot analyses of yeast lysates co-transformed with NSP4 or NSP4 mutants with caveolin-1 fusion proteins. The presence of NSP4- and cav-1 fusion proteins in the Y2H assay was shown by Western blot analyses of the yeast co-transformed with pD22cav-1 and each of the following fusion proteins: pD32NSP4 46-175 (lanes 2A and 2B), pD32NSP4-Ala6 (lanes 3A and 3B) , pD32NSP4-HydroMut (lane 4), pD32Rev113I (lane 5), pD32Rev124V (lane 6), and pD32Rev131Y (lane 7). Lanes 8 and 9 show the FLNSP4-HydroMut and FLNSP4, respectively. Blots were probed using (A) rabbit anti-NSP4150-175 or (B) rabbit anti-cav-12-31. Panel A: The non-transformed yeast (MaV203, lane 1) showed a non-specific band at ~29 kD that was observed in all lanes. Lysates expressing FLNSP4-HydroMut (lane 8) demonstrated NSP4-specific bands at ~ 28, 24 kD. Lysates expressing FLNSP4 showed bands at ~ 28, 24, and 20 kD (lane 9). All of the co-transformed yeast demonstrated a NSP4-fusion protein band at ~ 34.4 kD (A, lanes 2-7). Panel B revealed a faint contaminating band below the 34.9 kD specific caveolin-1-fusion protein band (lanes 1-7). Lanes 2-7 show the specific caveolin-1 fusion protein band at ~34.9 kD. Panel C: Lysates expressing FLNSP4 (lane 1) showed bands at ~ 24 and 28 kD and (lane 9). All of the co-transformed yeast demonstrated both monomeric and multimeric NSP4-specific fusion protein bands (lanes 1). Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed the ~34.9 kD specific NSP4-1-fusion protein band (lanes 2-5). Also, Rev1M124,127A demonstrated a multimeric NSP4-fusion specific band similar to one observed with FLNSP4. Panel D: Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed a ~ 34.9 kD specific caveolin-1-fusion protein band (lanes 1-4). Also, Rev1M124,127A demonstrated a multimeric caveolin-1-fusion protein band.
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Figure 3: Representative Western blot analyses of yeast lysates co-transformed with NSP4 or NSP4 mutants with caveolin-1 fusion proteins. The presence of NSP4- and cav-1 fusion proteins in the Y2H assay was shown by Western blot analyses of the yeast co-transformed with pD22cav-1 and each of the following fusion proteins: pD32NSP4 46-175 (lanes 2A and 2B), pD32NSP4-Ala6 (lanes 3A and 3B) , pD32NSP4-HydroMut (lane 4), pD32Rev113I (lane 5), pD32Rev124V (lane 6), and pD32Rev131Y (lane 7). Lanes 8 and 9 show the FLNSP4-HydroMut and FLNSP4, respectively. Blots were probed using (A) rabbit anti-NSP4150-175 or (B) rabbit anti-cav-12-31. Panel A: The non-transformed yeast (MaV203, lane 1) showed a non-specific band at ~29 kD that was observed in all lanes. Lysates expressing FLNSP4-HydroMut (lane 8) demonstrated NSP4-specific bands at ~ 28, 24 kD. Lysates expressing FLNSP4 showed bands at ~ 28, 24, and 20 kD (lane 9). All of the co-transformed yeast demonstrated a NSP4-fusion protein band at ~ 34.4 kD (A, lanes 2-7). Panel B revealed a faint contaminating band below the 34.9 kD specific caveolin-1-fusion protein band (lanes 1-7). Lanes 2-7 show the specific caveolin-1 fusion protein band at ~34.9 kD. Panel C: Lysates expressing FLNSP4 (lane 1) showed bands at ~ 24 and 28 kD and (lane 9). All of the co-transformed yeast demonstrated both monomeric and multimeric NSP4-specific fusion protein bands (lanes 1). Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed the ~34.9 kD specific NSP4-1-fusion protein band (lanes 2-5). Also, Rev1M124,127A demonstrated a multimeric NSP4-fusion specific band similar to one observed with FLNSP4. Panel D: Rev2M116A, Rev2M116,124,127A, Rev1,2M127A and Rev1M124,127A revealed a ~ 34.9 kD specific caveolin-1-fusion protein band (lanes 1-4). Also, Rev1M124,127A demonstrated a multimeric caveolin-1-fusion protein band.
Mentions: To verify the presence of the NSP4 and cav-1 GAL-4 fusion proteins in the yeast that showed no binding to cav-1 in the Y2H assay, the co-transformed lysates were probed with either anti-NSP4150-175 (Figure 3A and C) or anti-cav-12-31 (Figure 3B and D) peptide-specific antibodies. Controls included untransformed MaV203 yeast lysates. The controls failed to show an antibody-specific band at the correct molecular weight of the fusion proteins for both the activating domain (AD)-NSP4 and binding domain (BD)-cav-1. However, a non-specific band at ~28 kD was observed when blotted with both antisera (Figure 3, lane 1). All NSP4 and cav-1 fusion proteins tested expressed at the correct molecular weight (34.4 kD and 34.9 kD, respectively) and were reactive with NSP4- (Figure 3, panels A&C) and cav-1 peptide-specific antisera (panel B&D). These data established that the encoded sequences from both plasmids were translated as fusion proteins in the co-transformed yeast, even when no reactivity was detected.

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