Conformational Differences Unfold a Wide Range of Enterotoxigenic Abilities Exhibited by rNSP4 Peptides from Different Rotavirus Strains

Abstract

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.

Keywords: NSP4; Rotavirus diarrhea; diarrheal dose 50 (DD50); multimerization; nonstructural protein 4; thioflavin T; viral enterotoxin.; virulence.

Fig. (1)

(A). Schematic representation of the structural organization of rotavirus NSP4. H1, H2 and H3, N-terminal hydrophobic domains; ECM, Extra Cellular Matrix; ISVD, Interspecies variable domain; DLP, Double-layered particle, (B). Amino acid sequence alignment of the NSP4ΔN72 region from different rotavirus strains used in this study. E158K and K158E represent the mutant NSP4 proteins generated by site-directed mutagenesis using PCR from the symptomatic (N136) and asymptomatic (2KD/851) Vellore strains, respectively.

Fig. (2)

Tricine-SDS-PAGE of NSP4ΔN72 and ΔN112 polypeptides from different strains. 2A, Lanes 1-16 represent NSP4 of SA11, RRV, Hg18, NCDV, FRV99, EHP, EC, Wa, S2, ST3, 1040, I321, 116E, N136, 2KD/851 strains and N136-E158K mutant protein. Note that analysis of ΔN72 from only 15 strains and one mutant protein is shown in the figure. Faint bands corresponding to dimmer (D) and tetramer (T) are shown by arrows. 2B, NSP4ΔN112 peptides from 6 different strains. M, Pre-stained MW markers (Biorad).

Fig. (3)

ThT fluorescence spectra of different NSP4ΔN72 proteins. The figure insert shows the ThT fluorescence spectra of NSP4ΔN72 from the Vellore symptomatic (N136) and asymptomatic (2KD/851) strains and E158K mutant protein generated from N136. Note that the fluorescence spectrum of the mutant protein is identical to that from the asymptomatic strain 2KD/851.

Fig. (4)

Relative trypsin Resistance of NSP4ΔN72 proteins from different strains. The relative resistance to trypsin of the region from residue 72-146 among the different ΔN72 peptides is indicated on a scale of 0-100. M, Top two panels- Prestained MW markers (Biorad), bottom three panels- Low MW range markers (GIBCO-BRL).

Fig. (5)

Secondary structure analysis of recombinant NSP4ΔN72 proteins by Far UV-CD spectroscopy. Purified NSP4s exhibit coiled coil structures as revealed by the highly negative CD spectral values but with significant secondary structural differences among themselves.