The rotavirus non-structural protein NSP4 functions as the viral enterotoxin and intracellular receptor for the double-layered particles (DLP). The full-length protein cannot be expressed and/ or purified to homogeneity from bacterial or insect cells. However, a bacterially expressed and purified mutant lacking the N-terminal 72 aa (DN72) was recently obtained from strains Hg18 and SA11 exhibiting approximately 17-20-, 150-200-and 13166-15800-fold lower DD 50 (50% diarrhoea-inducing dose) values in suckling mice compared with that reported for the partially pure, full-length protein, a C-terminal M175I mutant and a synthetic peptide comprising aa 114-135, respectively, suggesting the requirement for a unique conformation for optimal functions of the purified protein. The stretch of approximately 40 aa from the C terminus of the cytoplasmic tail of the endoplasmic reticulum-anchored NSP4 is highly flexible and exhibits high sequence variation compared with the other regions, the significance of which in diarrhoea induction remain unresolved. Here, it was shown that every amino acid substitution or deletion in the flexible C terminus resulted in altered conformation, multimerization, trypsin resistance and thioflavin T (ThT) binding, and affected DLP binding and the diarrhoea-inducing ability of the highly diarrhoeagenic SA11 and Hg18 DN72 in suckling mice. These studies further revealed that high ThT fluorescence correlated with efficient diarrhoea induction, suggesting the importance of an optimal ThT-recognizable conformation in diarrhoea induction by purified NSP4. These results based on biological properties provide a possible conformational basis for understanding the influence of primary sequence variations on diarrhoea induction in newborn mice by purified NSP4s that cannot be explained by extensive sequence analyses.
NSP4 has been recognized as the rotavirus-encoded enterotoxin. However, a few studies failed to support its diarrheagenic activity. As recombinant NSP4 (rNSP4) peptides of different lengths were used in the limited number of studies, a comparison of relative diarrheagenic potential of NSP4 from different strains could not be possible. To better understand the diarrheagenic potential of NSP4 from different strains, in this report we have evaluated the enterotoxigenic activity of the deletion mutant ΔN72 that lacks the N-terminal 72 residues and the biologically relevant ΔN112 peptide which when derived from SA11 rotavirus strain were previously shown to be highly diarrheagenic in newborn mice. Detailed comparative analysis of biochemical and biophysical properties and diarrheagenic activity of the recombinant ΔN72 peptides from seventeen different strains under identical conditions revealed wide differences among themselves in their resistance to trypsin cleavage, thioflavin T (ThT) binding, multimerization and conformation without any correlation with their diarrhea inducing abilities. These results support our previously proposed concept for the requirement of a unique conformation for optimal biological functions conferred by cooperation between the N- and C-terminal regions of the cytoplasmic tail.
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