The rotavirus strains, their G and P serotype/genotype associations and the host from which they were isolated are listed in Table 1. The Vellore neonatal strains were kindly provided by Dr. G. Kang, Christian Medical College, Vellore, India. Except for IS2, 1040, EHP, EC and Vellore strains, viruses were grown in MA104 cells.

Table 2..

Comparative Analysis of Biochemical, Biophysical and Biological Properties of NSP4ΔN72 Polypeptides from 17 Different Rotavirus Strains

DD₅₀ values of NSP4ΔN72 and ΔN112 polypeptides from different rotavirus strains from different species in newborn mice are shown. The DD₅₀ values previously reported by others for different NSP4 peptides are also shown. The relative ThT fluorescence emission values are with reference to the lowest-binding ΔN72 from strain 1040. The conformational contents, relative resistance to trypsin digestion, multimerizatio/oligomerization properties of different NSP4ΔN72 peptides are provided. Note the asymptomatic strains from which the NSP4 is derived for this study are indicated in yellow shade. The level of efficiency of the proteins in each of the properties is classified into 4 grades in different colour shades. Pink colour refers to either highest efficiency or highest values. Green, gray and red shades denote the decreasing levels of efficiency or the values in the properties. Though efficient diarrhea inducers exhibit high ThT fluorescence, ∝-helical content, trypsin resistance and efficient multimerization, note a general lack of correlation among the properties of other proteins. FL, full length, ND, Not Determined, OSUv and OSUa refer to OSU virulent and avirulent strains, respectively. E158K and K158E mutant proteins are described in the legend to Fig. (1B).

The genomic RNA from EC, EHP, IS2, 1040, I321 and Vellore strains was extracted from the fecal samples and that from others was isolated from infected cell culture supernatants as described previously [36, 43]. Cloning of the NSP4 gene, its ΔN72 region spanning aa 73 to 175 and generation of the pET22-NH vector for expression of proteins in fusion with an N-terminal His-tag have been described [3]. The ΔN112 region was cloned using strain- and position-specific primers. The nucleotide sequence of ΔN72 and ΔN112 in pBluescript KS+ (pBS) or pET22-NH was determined using T7, M13 forward and/or reverse primers (Macrogen, Korea). All NSP4ΔN72 and ΔN112 peptides expressed in E. coli BL21 (DE3) were highly soluble and were purified by Ni²⁺-NTA-agarose (QIAGEN) chromatography after binding in presence 0.5% NP-40 and washing extensively in its absence [3]. Purity of the proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Molecular masses of the peptides were determined by size exclusion chromatography (SEC) using Sephacryl S-200 column (GE Healthcare) on a Bio-Rad FPLC chromatography system as well as by mass spectrometry using Ultraflex time of flight mass spectrometer (Bruker Daltonics) [3, 46].

ΔN72 peptides dialyzed against a buffer containing 10 mM sodium phosphate pH 7.6 and 100 mM NaCl at 100 μM each of the protein and the dye were used. ThT-binding assays were performed by mixing 50 µl of 60 µM protein solution with 450 µl of 10 µM ThT. Readings were recorded in a Shimadzu RF-5301 PC spectrofluorometer at 25°C. The excitation wave length was 450 nm, and the emission was monitored between 450 and 600 nm [3, 47].

Prior to animal experiments, the ThT binding ability of the ΔN72 peptides was evaluated [3]. Peptides exhibiting high ThT fluorescence were tested between 1 and 100 pmol and those showing highly reduced or lack of ThT binding were evaluated between 50 pmol and 10 nmol in 5-7 day-old BALB/c mouse pups. The recombinant ΔN72 and ΔN112 peptides in 50 μl of sterile PBS were administered intraperitoneally. DD₅₀ and mean diarrheal scores, on a scale of 1-4, were determined as described [2, 3]. At each dose, 8 mouse pups were used and the experiment was repeated three to four times. The fold efficiency of diarrhea induction of different NSP4ΔN72 peptides was calculated with reference to the DD₅₀ of SA11ΔN72 which exhibited the lowest value.

Secondary structural differences among different ΔN72 and ΔN112 peptides were examined employing Far UV-CD spectroscopy. The percent α-helical, β-sheet and random conformation contents were determined using the k2d program [48]. CD spectra of the proteins, in 5 mM sodium phosphate buffer pH 7.4 containing 5 mM NaCl, were recorded on a JASCO J-715 spectropolarimeter at a protein concentration of 10 μM and the molar residue ellipticity was calculated as described [3].

Purified ΔN72 peptides were digested with sequencing grade trypsin (Promega) at 37°C, the trypsin-cleaved products were analyzed by Tricine-SDS-PAGE [49] followed by Coomassie Blue staining and the molecular masses of the cleaved products were determined by mass spectrometry as previously described [3]. Relative trypsin resistance on a scale of 0 to 100% was determined by densitometric measurement of intensities of bands corresponding to all the protected fragments of the helical region post 2 hr incubation with respect to control peptide with 75 to 100% being highly resistant and 0 to 25% corresponding to undetectable level of protected fragments.

Recently, we have shown that rNSP4ΔN72 peptides from SA11 and Hg18 were highly diarrheagenic, formed highly ordered higher molecular weight (HMW) complexes, exhibited high α-helical content, thioflavin T binding and resistance to trypsin of the region from residue 73-146 in contrast to a large number of their N- and C-terminal deletion mutants or amino acid (aa) substitution mutants [3, 6]. Mutations in ΔN72 affected the diarrhea-inducing and ThT-binding activities to different extents with DD₅₀ increases ranging between 20-2080-fold (DD₅₀ 0.05 to >10 nmol) [3, 6]. These studies suggested a correlation between optimal ThT binding and efficient diarrhea induction and that efficient ThT binding is dependent on a unique conformation that is significantly affected by aa substitutions throughout the length of the peptide. Further, a specific conformation only in the ordered multimeric forms of SA11- and Hg18-NSP4ΔN72, but not other HMW complexes of mutant NSP4ΔN72 peptides, is recognized by ThT. Since NSP4s from different strains exhibit significant aa variations in the flexible C-terminal region of the cytoplasmic tail (CT) (Fig. 1B), it is likely that different NSP4 peptides would differ significantly from each other in their ThT binding property.

The observation that high ThT fluorescence exhibited by SA11- and Hg-18ΔN72 peptides correlated with their efficient diarrhea-inducing ability suggested that this property can be used to significantly reduce the number of mouse pups required to determine the DD₅₀ of the large number of NSP4 peptides used in this study. Thus a protein showing high ThT binding need not be tested at high concentration and those that exhibit weak binding can be tested only at high concentration.

The ΔN72 peptides from 17 different human (sympto-matic and asymptomatic) and animal strains (Table 1) were expressed, purified (Fig. 2A) and their ThT binding property was evaluated. As shown in Fig. (3), only I321-, SA11-, Hg18- and EHP- NSP4ΔN72 peptides exhibited significant ThT fluorescence and those from all other strains showed either highly reduced or total lack of ThT binding. Of note, whilst NSP4 from the human asymptomatic strain I321 showed about 2.0-3.3-fold more ThT fluorescence than that of Hg18 and SA11, that from other human asymptomatic strains116E and ST3 exhibited highly reduced or negligible ThT fluorescence. The peptides from the two murine strains EHP and EC also varied significantly in their ThT binding ability. Thus in contrast to the previous observation with SA11- and Hg18- NSP4ΔN72 peptides which exhibited high level of ThT binding, the present analysis using similar peptides from a large number of strains revealed that the ThT binding ability varied widely among different peptides.

Evaluation of the diarrhea inducing ability of ΔN72 peptides from 17 different strains suggested that different NSP4ΔN72 peptides used in this study can be identified as either efficient diarrhea inducers represented by SA11, Hg18 and EHP which exhibited a low DD₅₀ of 0.005-0.05 nmol, or inefficient/poor diarrhea inducers represented by other NSP4s exhibiting DD₅₀ > 0.05 nmol (Table 2). Although the efficient diarrhea-inducing activity of the former group correlated, in general, with high ThT fluorescence and spontaneous diarrhea within 30 minutes of administration, the inefficient diarrhea inducers differed widely among themselves without any correspondence between ThT fluorescence and DD₅₀ (Table 2). Further, NSP4s from symptomatic and asymptomatic strains could not be distinguished by their diarrhea inducing abilities. While the NSP4 peptides from human asymptomatic strains ST3, I321, and 116E exhibited DD₅₀ values between 0.07 and 0.5 nmol, those from animal strains and human symptomatic strains (RRV, NCDV, FRV18, EC, and Wa, IS2 and 1040) showed DD₅₀ in the range of 0.75 to >10 nmol. Also, the ΔN72 peptides from the Vellore symptomatic strains (N136 and N138) were about 33-fold less efficient in their ability to induce diarrhea in newborn mouse pups than those from the asymptomatic strains 2KD/851 and 99-D/214. Of note, I321 NSP4 which bound ThT better than SA11 and Hg18 NSP4ΔN72 peptides, was about 1000-fold less efficient in diarrhea induction (Table 2). Whilst ΔN72 from SA11, Hg18 and EHP induced spontaneous diarrhea within 30-40 min of protein administration at the DD₅₀ value with a mean diarrheal score of 3.2, the inefficient diarrheagenic peptides induced diarrheal stools at or below DD₅₀ values between 1 and 2 hr post administration only after gentle palpitation of the abdomen with mean diarrhoeal score of 2.0. Among the latter group, NSP4 from ST3 and NCDV exhibited comparatively lower DD₅₀ (0.07 and 0.075 nmol, respectively). While the peptides from RRV, Wa, equine strain FRV99 and the G2 strain 1040 showed 100-200-fold higher DD₅₀, that from another G2 strain IS2 exhibited 1000-fold higher DD₅₀ than SA11-NSP4ΔN72 (Table 2). Of significance, the murine EC-NSP4ΔN72 failed to induce diarrhea even with 10 nmol of the protein (precise DD₅₀ not determined) compared to that of another murine strain EHP (0.05 nmol). Though IS2-NSP4ΔN72 peptide was very inefficient in diarrheagenic activity (5.0 nmol) among the diarrheagenic ΔN72 peptides, it appears to be significantly better than the full-length proteins from avian strains (41-138 nmol) (Table 2) [23, 39].

Though ΔN72 from SA11 and Hg18 was very efficient in diarrhea induction, this mutant form of the protein is not detected in the virus infected cells. It is possible that the wide differences observed in the diarrheagenic properties of ΔN72 peptides from different strains could be a manifestation of the unnatural mutation. Since the full-length protein could not be expressed in E. coli and that expressed in insect cells was difficult to purify to homogeneity, we expressed and purified the recombinant ΔN112 peptide (Fig. 2B), which is similar to the biologically active peptide secreted from the virus infected or gene-transfected cells [14], from six different strains (SA11, Hg18, NCDV, I321, EHP and Wa) and determined their DD₅₀ in newborn mouse pups. As shown in Table 2, though the pattern of diarrhea inducing ability of the ΔN112 peptides from SA11, Hg18, NCDV, Wa and I321 was very similar to that of the corresponding ΔN72 peptides, the former exhibited high DD₅₀ values than the latter. These results suggest that both ΔN72 and ΔN112 peptides from different strains exhibit very similar pattern in their diarrhea inducing properties, but differ in their relative diarrhea inducing efficiencies. The relatively high DD₅₀ values exhibited by the ΔN112 peptides in comparison to those of ΔN72 peptides could be attributed to their lack of the N-terminal amphipathic domain which was shown to potentiate the biological function of the protein [3, 20].

Recently, G10P [11] strains associated with symptomatic or asymptomatic infections in neonates in Vellore, India, were reported. However, sequence analysis of NSP4 from a few isolates failed to differentiate the virulent from the avirulent strains [45]. Sequence analysis, in our laboratory, of NSP4ΔN72 from two symptomatic Vellore isolates (N136 and N138) for which the sequence was not reported, and two asymptomatic isolates (2KD/851 and 99-D/214) for which the sequence was available, revealed a single aa difference at position 158 (E158K) between the virulent and avirulent pairs. While the symptomatic isolates contained Glu at this position, the asymptomatic strains possessed Lys (Fig. 1B). Only Glu was reported at position 158 in both symptomatic and asymptomatic strains [45]. To understand if the virulence phenotype of the Vellore strains correlates with the diarrheagenic activity of cognate NSP4, the DD₅₀ of ΔN72 peptides from symptomatic and asymptomatic isolates in suckling mice was determined. Unexpectedly, NSP4ΔN72 peptide from the symptomatic isolates N136 and N138 exhibited DD₅₀ (2.0 nmol) that was about 33-fold higher than that of the asymptomatic isolates 2KD/851 and 99-D/214 (0.06 nmol). Association of the E158K mutation with altered diarrhea induction was further evident from the DD₅₀ values of the E158K and K158E mutant peptides which were indistinguishable from those of the peptides from asymptomatic and symptomatic strains, respectively (Table 2). Of note, N136-ΔN72 consistently showed >50% higher ThT fluorescence of that of 2KD/851, though significantly less than that showed by SA11 and Hg18, (Fig. 3) but was relatively inefficient in diarrhea induction than 2KD/851.

Though ThT is frequently used to detect β-sheet structures in amyloid fibrils and ordered polymeric proteins [50, 51], studies on acetylcholinesterase [52] revealed that ThT binds efficiently to the peripheral ligand binding site which lacks β-sheet structures characteristic of amyloid protein. Though the precise nature of interaction is not understood, these as well as our results [3, 6] suggested that ThT could bind to proteins independent of the amyloid β-sheet structures and that different amino acid substitution and deletion mutant NSP4ΔN72 peptides differ significantly in their conformation.

The ΔN72 peptide region from different strains contains 17 trypsin cleavage sites that are conserved among the strains (Fig. 1B). Previously, we have shown that single amino acid substitutions in the CT of the highly diarrheagenic SA11-NSP4ΔN72 affected its diarrhea inducing ability and resistance to trypsin digestion [3]. Since different NSP4s significantly differ in their sequence in the unstructured C-terminal region, it is likely that NSP4s from different strains would vary in their susceptibility to trypsin cleavage. As shown in Fig. (4), the order of trypsin resistance on a scale of 0 to 100 (100 being the highest) was SA11, Hg18, Wa, EHP, EC, 2KD/851 and N136-E158K (75-100%) > ST3, RRV and N136 (50 to 75%) > IS2, I321, 116E (25 to 50%) > NCDV and 1040 (0 to 25%), with the SA11 group being highly resistant and NCDV group being extremely susceptible. While ΔN72 from SA11, Hg18 and WA yielded a 9.95 kDa stable fragment, that from the murine strains yielded additional smaller stable fragments (Fig. 4). Further, the non-diarrheagenic EC-NSP4ΔN72 was as resistant to trypsin as EHP-NSP4ΔN72. It may be noted that NSP4s from the murine strains exhibit high sequence divergence compared to that of other group A rotaviruses and form a distinct genetic group [22] (Fig. 1B). Of significance, ΔN72 from the symptomatic strain N136 was highly susceptible to trypsin and about 33-fold less diarrheagenic compared to that from the asymptomatic strain 2KD/851 or the N136-E158K mutant in spite of the protein from N136 exhibiting relatively higher ThT fluorescence (Figs. 3, 4 and Table 2).

The wide differences in ThT binding and susceptibility to trypsin digestion suggest conformational differences among the recombinant ΔN72 peptides from different strains. CD spectroscopic studies were employed to confirm this prediction. As shown in Fig. (5) and Table 2, all the peptides exhibited highly negative CD spectral values as expected suggesting that they contained the expected coiled coil domain (CCD) and the highly diarrheagenic SA11-, Hg18- and EHP-ΔN72 peptides consistently showed high (-helical content compared to majority of the peptides as observed earlier [3]. Thus while high (-helical content correlated well with the low DD₅₀ values of the efficient diarrhea inducers, the inefficient diarrhea inducers differed significantly among themselves in their conformation contents without correlation to their relative DD₅₀ values. For example, while NSP4ΔN72 from Wa, ST3, 116E, N136 and N138 exhibited about 56-58%, that from all other strains showed only 30-48% (-helical content. Generally, proteins exhibiting high (-helicity contained less than 9% (-sheet content and those with low (-helical content showed between 16 and 26%. NCDV-NSP4ΔN72 differed from all others as it failed to bind ThT and exhibited very high (54%) random conformation content in contrast to that of other proteins which ranged between 32 and 44%. The SA11ΔN112 peptide contained very low (-helical content compared to the corresponding ΔN72 peptide. Hence the secondary structural contents of ΔN112 peptides from other strains were not evaluated.

Conformational differences appeared to reflect the ordered multimerization ability of the different recombinant proteins. We recently showed that the highly diarrheagenic NSP4ΔN72 from SA11 and Hg18 existed in HMW complexes in E. coli, and that the purified protein formed highly ordered multimers and mutations in the N- or C-terminal regions significantly perturbed the conformation and equilibrium between the multimeric and oligomeric forms to different extents [3, 6]. As shown in Table 1, while the highly diarrheagenic SA11- and Hg18-ΔN72 peptides exhibited efficient multimerization even at low concentration as reported earlier [3], all other ΔN72 peptides differed widely in their multimerization property. Some showed concentration-dependent multimerization (EHP and RRV), whereas others either existed predominantly in oligomeric form or failed to multimerize even at high concentration (Table 2). Though the rNSP4ΔN72 from the symptomatic and asymptomatic Vellore strains differing in a single amino acid showed comparable multimerization/oligomerization properties, they differed significantly in their ThT binding, trypsin resistance and conformation contents. NCDV-NSP4ΔN72 though failed to multimerize at low concentration, about 60% existed as HMW form at high concentration. Interestingly, its diarrhea-inducing ability showed a sharp decline below DD₅₀ value, which probably correlates with the extreme instability of the oligomers at low concentration as seen by the broad peak in SEC (data not shown).