Rotaviruses, members of the family Reoviridae, are the major etiologic agents of severe, acute dehydrating diarrhea in the young of many mammalian species, including humans, calves and foals. Recent estimates indicate an annual death toll of approximately 600,000 infants due to rotavirus, besides inflicting staggering economic burden worldwide. Most of these deaths occur in the developing countries and India is estimated to account for about a quarter of these deaths. Extensive molecular epidemiology studies carried out by our laboratory have revealed many interesting aspects about rotavirus diversity in this country. Molecular epidemiology of rotaviruses causing severe diarrhea in foals in two organized farms in northern India was carried out. These foal rotaviruses exhibited 5 different electropherotypes (E), E1-E5. Strains belonging to E1, E2 and E5 exhibited G10, P6[1]; G3 and G1 type specificities. Though the E1 strains possessed genes encoding G10 and P6[1] type outer capsid proteins, unlike the G10, P8[11] type strain I321, they exhibited high reactivity with the G6-specific MAb suggesting that the uncommon combination altered the specificity of the conformation-dependent antigenic epitopes on the surface proteins. Strains belonging to electropherotypes E3 and E4 were untypeable. Sequence analysis of the VP7 gene from E4 strains (Erv92 and Erv99), revealed that they represent a new VP7 genotype, G16. Nonstructural protein 4 (NSP4) of rotavirus is a multidomainal, multifunctional protein and is the first viral enterotoxin identified. We have recently reported that the diarrhea-inducing and double-layered particle (DLP)–binding properties of NSP4 are dependent on a structurally and functionally overlapping conformational domain that is conferred by cooperation between the N- and C-terminal regions of the cytoplasmic tail (Jagannath et al., J. Virol, pp 412-425, 2006). Further, a stretch of 40 amino acids (aa) from the C-terminus is predicted to be unstructured and highly susceptible to trypsin cleavage. We examined the role of this unstructured C-terminus of Hg18 NSP4 and SA11 NSP4 on the biological properties of NSP4 using a series of deletion and substitution mutants of the conserved proline and tyrosine residues in this region. Gel filtration, CD spectroscopy and Thioflavin T binding studies showed that these mutants have altered secondary structural contents and either failed to multimerize efficiently or multimerized with altered conformation. The C-terminal ten residues appear to play a regulatory role on multimerization. Proline 168, tyrosine 166 and methionine 175 appear to be critical determinants of DLP binding activity whereas, proline 165 and tyrosine 85 and 131 appears to determine the affinity of binding to DLP in the context of NSP4 ∆N72. Deletion and substitution mutants exhibited severely reduced diarrhea inducing ability and DLP binding property. Of great biological significance is the drastic decrease in the diarrhea inducing ability of the N- and C- terminal deletion mutant ∆N94 ∆C29 that exhibited about 11,000-fold increase in DD50 than the wild type (WT) ∆N72. These studies revealed that the predicted unstructured C-terminus is an important determinant of biological properties of NSP4. Extensive efforts to crystallize the complete cytoplasmic tail (CT) of NSP4 were unsuccessful and to date, the structure of only a synthetic peptide corresponding to aa 95-135 has been reported. Our recent studies indicate that the interspecies variable regions from aa 135-141 as well as the extreme C-terminus are critical determinants of virus virulence and diarrhea-inducing ability of the protein. Here, we examined the crystallization properties of several deletion mutants and report the structure of a mutant recombinant NSP4 from symptomatic (SA11) and asymptomatic (I321) strains that lacked the N-terminal 94 and C-terminal 29 aa (NSP4: 95-146) at 1.67 Å and 2.7Å, respectively. In spite of the high-resolution data, electron density for the stretch of 9 residues from the C-terminus could not be seen suggesting its highly flexible nature. The crystal packing showed a clear empty space for this region. Extension of the unstructured C-terminus beyond aa 146 hindered crystallization under the experimental conditions. The present structure revealed significant differences from that of the synthetic peptide in the conformation of amino acids at the end of the helix as well as crystal packing owing to the additional space required to accommodate the unstructured virulence-determining region. Conformational differences in this critical region effected by the presence or absence of proline or glycine at specific positions in the unstructured C-terminus, could form the basis for the wide range of variation seen in the diarrhea-inducing ability of NSP4 from different strains in newborn mouse pups. Although symptomatic and asymptomatic strains do not generally differ in the presence or absence of the conserved prolines or glycines, they contain a few additional changes that could alter the unique conformation required for optimal biological activity. In conclusion, we demonstrate that the predicted unstructured C-terminal region is indeed highly flexible and is an important determinant of biological functions of the NSP4, mutations in which probably correlates with the virulence properties of the virus.