Rotavirus is the major cause of dehydrating gastroenteritis in children and young animals. NSP4 (non-structural protein 4), a rotaviral non-structural glycoprotein and a peptide NSP4114–135 (DKLTTREIEQVELLKRIYDKLT), corresponding to NSP4 amino acids 114–135, induce diarrhoeal disease in a neonatal mouse model and interact with model membranes that mimic caveolae. Correlation of the mechanisms of diarrhoea induction and membrane interactions by NSP4 protein and peptide remain unclear. Several additional NSP4 peptides were synthesized and their interactions with membranes studied by (i) CD, (ii) a filtration-binding assay and (iii) a fluorescent molecule leakage assay. Model membranes that varied in lipid compositions and radius of curvature were utilized to determine the compositional and structural requirements for optimal interaction with the peptides of NSP4. Similar to the intact protein and NSP4114–135, peptides overlapping residues 114–135 had significantly higher affinities to membranes rich in negatively charged lipids, rich in cholesterol and with a high radius of curvature. In the leakage assay, small and large unilamellar vesicles loaded with the fluorophore/quencher pair 8-aminonaphthalene-1,3,6-trisulphonic acid disodium salt/p-xylene-bis-pyridinium bromide were incubated with the NSP4 peptides and monitored for membrane disruption by lipid reorganization or by pore formation. At a peptide concentration of 15 µM, none of the NSP4 peptides caused leakage. These results confirm that NSP4 interacts with caveolae-like membranes and the α-helical region of NSP4114–135 comprises a membrane interaction domain that does not induce membrane disruption at physiological concentrations.
- liposome leakage
- peptide—membrane interaction
- rotavirus non-structural protein 4 (NSP4)
Abbreviations used: ANTS, 8-aminonaphthalene-1,3,6-trisulphonic acid disodium salt; CFTR, cystic fibrosis transmembrane conductance regulator; DOPS, 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine]; DPX, p-xylene-bis-pyridinium bromide; ER, endoplasmic reticulum; LUV, large unilamellar vesicles; NSP4, non-structural protein 4; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PS, phosphatidylserine; SUV, small unilamellar vesicles; TFE, trifluoroethanol; TNBS, 2,4,6-trinitrobenzenesulphonic acid.
- The Biochemical Society, London ©2004