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The flexible C terminus of the rotavirus non-structural protein NSP4 is an important determinant of its biological properties

¹ Department of Microbiology & Cell Biology, Indian Institute of Science, Bangalore 560012, India
² Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India

Correspondence
C. Durga Rao
cdr{at}mcbl.iisc.ernet.in

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 (N72) was recently obtained from strains Hg18 and SA11 exhibiting approximately 17–20-, 150–200- and 13166–15800-fold lower DD₅₀ (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 N72 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.

The oligonucleotides used in this study are presented as a supplementary table available with the online version of this paper.