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Elucidation of the Rotavirus NSP4-Caveolin-1 and -Cholesterol Interactions Using Synthetic Peptides.

Schroeder ME, Hostetler HA, Schroeder F, Ball JM - J Amino Acids (2012)

Bottom Line: NSP4(112-140) that overlaps the caveolin-1 binding domain and a cholesterol recognition amino acid consensus (CRAC) motif and both termini of caveolin-1 (N-caveolin-1(2-20),  (19-40) and C-caveolin-1(161-180)) were synthesized.Intracellular cholesterol alteration revealed a redistribution of NSP4 and disintegration of viroplasms.These data further imply interruption of NSP4(112-140)-N-caveolin-1(19-40) and cholesterol interactions may block NSP4 intracellular transport, hence enterotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathobiology, Texas A&M University, TVMC, College Station, TX 77843-4467, USA.

ABSTRACT
Rotavirus (RV) NSP4, the first described viral enterotoxin, is a multifunctional glycoprotein that contributes to viral pathogenesis, morphogenesis, and replication. NSP4 binds both termini of caveolin-1 and is isolated from caveolae fractions that are rich in anionic phospholipids and cholesterol. These interactions indicate that cholesterol/caveolin-1 plays a role in NSP4 transport to the cell surface, which is essential to its enterotoxic activity. Synthetic peptides were utilized to identify target(s) of intervention by exploring the NSP4-caveolin-1 and -cholesterol interactions. NSP4(112-140) that overlaps the caveolin-1 binding domain and a cholesterol recognition amino acid consensus (CRAC) motif and both termini of caveolin-1 (N-caveolin-1(2-20),  (19-40) and C-caveolin-1(161-180)) were synthesized. Direct fluorescence-binding assays were employed to determine binding affinities of the NSP4-caveolin-1 peptides and cholesterol. Intracellular cholesterol alteration revealed a redistribution of NSP4 and disintegration of viroplasms. These data further imply interruption of NSP4(112-140)-N-caveolin-1(19-40) and cholesterol interactions may block NSP4 intracellular transport, hence enterotoxicity.

No MeSH data available.


Related in: MedlinePlus

Direct Fluorescence-Binding Assays of N-Cav2-20 or N-Cav19-40 Peptide with C-Cav161-178 Peptide. (a) Fluorescence spectra of Cy3-C-Cav161-178 titrated with increasing concentrations of Cav19-40 (0–500 nM). (b-c) Plots of maximal fluorescence emission (measured at 565 nm upon excitation at 550 nm) for (b) N-Cav2-20 and (c) N-Cav19-40 in the presence of 100 nM Cy3-C-Cav161-178. Insets: linear plots of the binding curve of (b) N-Cav2-20 and (c) N-Cav19-40.
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fig3: Direct Fluorescence-Binding Assays of N-Cav2-20 or N-Cav19-40 Peptide with C-Cav161-178 Peptide. (a) Fluorescence spectra of Cy3-C-Cav161-178 titrated with increasing concentrations of Cav19-40 (0–500 nM). (b-c) Plots of maximal fluorescence emission (measured at 565 nm upon excitation at 550 nm) for (b) N-Cav2-20 and (c) N-Cav19-40 in the presence of 100 nM Cy3-C-Cav161-178. Insets: linear plots of the binding curve of (b) N-Cav2-20 and (c) N-Cav19-40.

Mentions: To evaluate the mechanism of the interaction of NSP4 with both cav-1 termini, we examined whether the two cav-1 termini bound one another to facilitate the interaction with NSP4 by employing the fluorescence-binding assay. C-Cav161-178 was labeled with a Cy3-fluorophore and titrated with either the N-Cav2-20 or N-Cav19-40 peptide. Titration of C-Cav161-178 with increasing concentrations of either N-terminal peptide resulted in no change in fluorescence intensity at 565 nm, indicative of a lack of association (Figure 3). Hence, we propose that the N- and C- termini of cav-1 do not bind one another and the interaction between NSP4 and the cav-1 termini does not result from an initial binding between the cav-1 N- and C-termini. To our knowledge, this is the first report of the cav-1 termini not interacting. Additional studies are needed to dissect the mechanism and implications of both cav-1 termini binding NSP4.


Elucidation of the Rotavirus NSP4-Caveolin-1 and -Cholesterol Interactions Using Synthetic Peptides.

Schroeder ME, Hostetler HA, Schroeder F, Ball JM - J Amino Acids (2012)

Direct Fluorescence-Binding Assays of N-Cav2-20 or N-Cav19-40 Peptide with C-Cav161-178 Peptide. (a) Fluorescence spectra of Cy3-C-Cav161-178 titrated with increasing concentrations of Cav19-40 (0–500 nM). (b-c) Plots of maximal fluorescence emission (measured at 565 nm upon excitation at 550 nm) for (b) N-Cav2-20 and (c) N-Cav19-40 in the presence of 100 nM Cy3-C-Cav161-178. Insets: linear plots of the binding curve of (b) N-Cav2-20 and (c) N-Cav19-40.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303745&req=5

fig3: Direct Fluorescence-Binding Assays of N-Cav2-20 or N-Cav19-40 Peptide with C-Cav161-178 Peptide. (a) Fluorescence spectra of Cy3-C-Cav161-178 titrated with increasing concentrations of Cav19-40 (0–500 nM). (b-c) Plots of maximal fluorescence emission (measured at 565 nm upon excitation at 550 nm) for (b) N-Cav2-20 and (c) N-Cav19-40 in the presence of 100 nM Cy3-C-Cav161-178. Insets: linear plots of the binding curve of (b) N-Cav2-20 and (c) N-Cav19-40.
Mentions: To evaluate the mechanism of the interaction of NSP4 with both cav-1 termini, we examined whether the two cav-1 termini bound one another to facilitate the interaction with NSP4 by employing the fluorescence-binding assay. C-Cav161-178 was labeled with a Cy3-fluorophore and titrated with either the N-Cav2-20 or N-Cav19-40 peptide. Titration of C-Cav161-178 with increasing concentrations of either N-terminal peptide resulted in no change in fluorescence intensity at 565 nm, indicative of a lack of association (Figure 3). Hence, we propose that the N- and C- termini of cav-1 do not bind one another and the interaction between NSP4 and the cav-1 termini does not result from an initial binding between the cav-1 N- and C-termini. To our knowledge, this is the first report of the cav-1 termini not interacting. Additional studies are needed to dissect the mechanism and implications of both cav-1 termini binding NSP4.

Bottom Line: NSP4(112-140) that overlaps the caveolin-1 binding domain and a cholesterol recognition amino acid consensus (CRAC) motif and both termini of caveolin-1 (N-caveolin-1(2-20),  (19-40) and C-caveolin-1(161-180)) were synthesized.Intracellular cholesterol alteration revealed a redistribution of NSP4 and disintegration of viroplasms.These data further imply interruption of NSP4(112-140)-N-caveolin-1(19-40) and cholesterol interactions may block NSP4 intracellular transport, hence enterotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathobiology, Texas A&M University, TVMC, College Station, TX 77843-4467, USA.

ABSTRACT
Rotavirus (RV) NSP4, the first described viral enterotoxin, is a multifunctional glycoprotein that contributes to viral pathogenesis, morphogenesis, and replication. NSP4 binds both termini of caveolin-1 and is isolated from caveolae fractions that are rich in anionic phospholipids and cholesterol. These interactions indicate that cholesterol/caveolin-1 plays a role in NSP4 transport to the cell surface, which is essential to its enterotoxic activity. Synthetic peptides were utilized to identify target(s) of intervention by exploring the NSP4-caveolin-1 and -cholesterol interactions. NSP4(112-140) that overlaps the caveolin-1 binding domain and a cholesterol recognition amino acid consensus (CRAC) motif and both termini of caveolin-1 (N-caveolin-1(2-20),  (19-40) and C-caveolin-1(161-180)) were synthesized. Direct fluorescence-binding assays were employed to determine binding affinities of the NSP4-caveolin-1 peptides and cholesterol. Intracellular cholesterol alteration revealed a redistribution of NSP4 and disintegration of viroplasms. These data further imply interruption of NSP4(112-140)-N-caveolin-1(19-40) and cholesterol interactions may block NSP4 intracellular transport, hence enterotoxicity.

No MeSH data available.


Related in: MedlinePlus