Rotavirus is the major cause of infantile gastroenteritis. Each year more than 600,000 young children are estimated to die in developing countries throughout the world. Rotavirus infection can be either symptomatic or asymptomatic. But the genetic or molecular basis for rotavirus virulence is not yet clearly understood. NSP4, encoded by genome segment 10, is a multifunctional protein. It is identified as the first viral enterotoxin and is essential for virus morphogenesis and pathogenesis. Analysis of NSP4 from more than 175 strains failed to reveal any sequence motif or amino acid that segregated with the virulence phenotype of the virus. Further, a few studies indicated a lack of consistent correlation between virus virulence and diarrhea inducing ability of the cognate NSP4. To understand the basis for the inconsistency in the enterotoxigenic activity of a few NSP4s reported in a limited number of studies, comparative analysis of the biophysical, biochemical, and biological properties of NSP4ΔN72, which from SA11 and Hg18 was earlier shown to be highly diarrheagenic, from 17 different symptomatic and asymptomatic strains was carried out. To study structure-function relationship we used Thioflavin T fluorescence assay, gel filtration, CD spectroscopy, trypsin susceptibility and enterotoxin assay in newborn mice for all the proteins. Detailed comparative analysis of biochemical and biophysical properties and diarrheagenic activity of the recombinant ΔN72 peptides under identical conditions revealed wide differences among themselves in their resistance to trypsin cleavage, thoflavin T binding, multimerization and conformation without any correlation with their diarrhea inducing abilities. Since earlier studies showed that a secreted peptide (ΔN112) of SA11-NSP4 also induced diarrhea in newborn mice pups, we have generated NSP4ΔN112 deletions from six different strains and tested for their diarrhea inducing ability. The patterns of DD50 values of the ΔN112 peptides was similar to that for ΔN72 peptides, but were 1000-1200-fold less efficient than that of SA11ΔN72. NSP4 exists in multiple forms in the infected cells- as oligomers, higher molecular weight complexes and ER- and cytoplasmic membrane anchored forms. Previous studies suggest that the N-terminal boundary of the oligomerization domain could lie downstream to residue 94 from the N-terminus. A peptide from residue 112-175, secreted from rotavirus infected cells, was reported to induce dose-dependent diarrhea in suckling mice, suggesting that the N-terminal boundary of the enterotoxin activity could lie around residue 112. However, the precise N-terminal boundaries in NSP4 for oligomerization and diarrhea induction have not been identified. To address this question, a large number of deletion mutants C-terminal to residue 94 were generated and tested for their ability to induce diarrhea in newborn mouse pups. Our data suggest that while the deletions ∆N121 to ∆N131 failed to induce diarrhea, ΔN118 was diarrheagenic suggesting that the N-terminal boundary of the minimal diarrhea inducing domain lies between aa 118 and 121. Size exclusion chromatography revealed that residues 95 to 98 are critical and sufficient for oligomerization. Studies on oligomerization further revealed that NSP4ΔN94 exists in pentamers, tetramers and dimers, while deletion mutants C-terminal to aa 94 exist only as dimers. Our studies demonstrate for the first time that not only tetramers but pentamers as well as dimers possess enterotoxigenic properties. Most human rotavirus infections are caused by group A rotaviruses. Within this group, rotaviruses are further classified into subgroups based on the antigenic specificity associated with the protein product of the sixth gene, VP6. Previous studies have mapped SG I specificity to aa position 305 and the region between 296 and 299, and SG II specificity to residue 315 on VP6. However, the subgroup specific determinants on NSP4 have not been identified till date. In this study, we generated several amino acid substitution mutants in the SG I-specific SA11 NSP4∆N72 protein as in previous studies ∆N72 was found to efficiently bind DLPs. Using an enzyme linked immunosorbent assay method, the effect of the mutations in the C-terminal and N-terminal regions in ∆N72 on their binding ability to SG I and SG II DLPs was assayed. Residues at positions 85, 169, 174 and 175 and in the ISVD appear to collectively determine the specificity of binding to DLPs. While the conserved proline and glycines at positions 165, 168 and 162, respectively, are important for maintaining the required conformation for general recognition of DLP. The present study provides important insights towards understanding the determinants in NSP4 for SG-specific DLP interaction.

Structuraj investigations on the Ddi1 (DNA-damage inducible protein 1) of Leishmania major and on the rotavirus nonstructural protein NSP4 were carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs effectively controlled opportunistic diseases caused by many parasitic protozoa such as Leishmania and Plasmodium species. The retroviral protease-like domains present in Ddi1 proteins of these organisms were identified as the targets of these drugs. Structural studies on Ddi1 from L. major have been carried out, in an attempt to provide a platform for the design of anti-protozoal compounds. Rotavirus NSP4, the first viral enterotoxin to be identified, is a multifunctional glycoprotein that plays critical roles in viral pathogenesis and morphogenesis. As part of an ongoing project on the structural characterization of NSP4, we determined the structure of the diarrhea-inducing region of this protein from the rotavirus strain MF66. Chapter 1 presents an overview of Ddi1 and NSP4 of the rotavirus with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Structural studies on the retroviral protease-like domain of Ddi1 (Ddi1-RVP) of L. major is presented in Chapter 3. Apart from this domain, Ddi1 of L. major also has a ubiquitin-associated and ubiquitin-like domains whereas P. falciparum has only the ubiquitin-associated domain. Activity of the full length Ddi1 of L. major and the retroviral protease domain of P. falciparum using an HIV protease substrate was shown to be inhibited by an HIV protease inhibitor, saquinavir. Binding of saquinavir to the proteins was also confirmed by Biolayer Interferometry studies. The crystal structure of the retroviral protease domain of L. major Ddi1 has been determined. It forms a homodimeric structure similar to that of HIV protease and the reported structure of the same domain from Saccharomyces cerevisiae. The loops in Ddi1-RVP are similar to the 'flap' regions of the HIV protease which close-in upon substrate/inhibitor binding; they are visible in the electron density maps, unlike the case of the S. cerevisiae protein. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative movements of the subunits. The present structure of Ddi1-RVP of L. major with the defined 'flap'-like loops will be helpful in the design of effective drugs against protozoal diseases, starting with HIV protease inhibitors as the lead compounds. Chapter 4 describes the structural investigations carried out on the diarrhea-inducing region of the nonstructural protein NSP4 of the rotavirus strain MF66 which forms an α-helical coiled-coil structure. Crystal structures of a synthetic peptide and of two recombinant proteins spanning this region showed parallel tetrameric organization of this domain with a bound Ca2+ ion at the core. Subsequently, we determined the structure of NSP4 from a different strain as a pentamer without the bound Ca2+ ion. This new structure provides more insights into understanding some of the functions of NSP4 such as the release of ions into the cytoplasm and binding to the double-layered particle (DLP). We also established conditions responsible for these structural transitions. The crystal structure of the coiled-coil domain of NSP4 presented in this chapter shows an entirely different structure which is an antiparallel tetramer. This explains our failure to determine the structure by the molecular replacement method using known oligomers. The structure was solved by the Sulphur-SAD method using diffraction data collected with Cr Ka radiation. The study reveals that the structural diversity of NSP4 is not limited. We could relate sequence variations and pH conditions to the differences in oligomeric assemblies. Surface properties of the domain suggest that the new form is likely to interact with different sets of proteins compared to those that interact with the parallel tetramers or pentamers. Further investigations are needed to establish this property.

Rotavirus is one of the leading etiological agents of gastroenteritis in young of many species including humans worldwide and is responsible for about 600,000 infant deaths per annum. Rotavirus belongs to the Reoviridae family, and its genome is composed of 11 double-stranded RNA segments that encode six structural proteins and six nonstructural proteins. Rotavirus replication is fully cytoplasmic and occurs within highly specialized regions called viroplasms. NSP2 and NSP5 have been shown to be essential for viroplasm formation and, when co-expressed in uninfected cells, to form viroplasm¬like structures. A recent study suggest a key role for NSP5 in architectural assembly of viroplasms and in recruitment of viroplasmic proteins, containing four structural (VP1, VP2, VP3 and VP6) and two nonstructural (NSP2 and NSP5) proteins. NSP5, the translation product of gene segment 11 has a predicted molecular eight of 21 kDa. NSP5 has been reported to exist in multiple isoforms ranging in size from 28-and 32-35 kDa from a 26-kDa precursor has been attributed to O-glycosylation and hyperphosphorylation. To study different properties of the protein, recombinant NSP5 containing an N-terminal hisidine tag was expressed in bacteria and purified by affinity chromatography. A significant observation was the similarity in phosphorylation property of the bacterially expressed and that expressed in mammalian cells. While the untagged recombinant protein failed to undergo phosphorylation in vitro, addition of His tag or deletions at the N-terminus promoted phosphorylation of the protein in vitro, which is very similar to the reported properties exhibited by the corresponding proteins expressed in mammalian cells. Phosphorylation of NSP5 in vitro is independent of the cell type from which the extract is derived suggesting that the kinases that phosphorylate NSP5 are distributed in all cell types. Among the C-terminal deletion mutants studied, NH-∆C5 and NH-∆C10 were phosphorylated better than full-length NSP5, but NH-∆C25 and NH¬∆C35 showed substantial reduction in the level of phosphorylation compared to full-length NSP5. These results indicate that the C-terminal 30 residues spanning the predicted α-helical domain of NSP5 are critical for its phosphorylation in vitro which is in correspondence with previous findings that C-terminal 21 amino acids of NSP5 direct its insolubility, hyperphosphorylation, and VLS formation. The results revealed that though the tagged full-length and some of the mutants could be phosphorylated in vitro, they are not suitable substrates for hyperphosphorylation unlike the similar proteins expressed in mammalian cells or infected cells. Analysis by western blot and mass spectrometry revealed that the bacterially expressed NH-NSP5 is indeed phosphorylated. It appears that prior phosphorylation in bacteria renders the protein conformationally not amendable for hyperphosphorylation by cellular kinases in vitro. Mutation of the highly conserved proline marginally enhanced its phosphorylation in vitro but the stability of protein is affected. Notably, mutation of S67A, identified as a critical residue for the putative caesin kinase-I and-II pathways of NSP5 phosphorylation, affected neither the phosphorylation nor the ATPase activity of NSP5. These results suggest that bacterially expressed NSP5 by itself has undectable auto-kinase activity and it is hypophosphorylated. Purified recombinant NSP5 has been reported to possess an Mg¬ 2+-dependent ATP-specific triphosphatase activity. The results indicated that deletion of either C-terminal 48 amino acids or N-terminal 33 residues severely affected the ATPase activity of recombinant NSP5, underlying the importance of both N-and C-terminal domains for NSP5 ATP hydrolysis function. NSP5 expressed in rotavirus infected cells exists as inter-molecular disulfide-linked dimeric forms and it appears that the 46 kDa isoforms, that are phosphorylated, corresponds to dimer as revealed by western blotting. Analytical gel filtration analysis of NH-NSP5, NH-ΔN43 and NH-ΔN33-ΔC25 showed most of the proteins in void volume, but an additional peak corresponding to the mass of dimeric species further supports that NSP5 is basically a dimer that undergoes oligomerization. Analysis by sucrose-gradient fractionation revealed that NH-NSP5 and NH-ΔN43 proteins were mainly distributed in the lower fraction of the gradient suggesting the existence of high molecular weight complexes or higher oligomers. The multimeric nature of NSP5 and its mutants was further confirmed by dynamic light scattering which suggests that high molecular weight complexes are of homogenous species. The correlation curves showed a low polydispersity distribution and a globular nature of recombinant NH-NSP5 proteins. The present results clearly demonstrate that dimer is the basic structural unit of NSP5 which undergoes oligomerization to form a complex consisting of about 20-21 dimers. The nonstructural protein 5 is hyperphosphorylated in infected cells and cellular kinases have been implicated to be involved in its phosphorylation. NSP5 contains multiple consensus sites for phosphorylation by several kinases, but the cellular kinases that specifically phosphorylate NSP5 in infected cells are yet to be identified. Previous studies from our laboratory using signaling pathway inhibitors revealed that recombinant NH¬NSP5 and its deletion mutants can be phosphorylated in vitro by purified cellular kinases and by mammalian cell extracts. These studies also showed the involvement of PI3K-Akt and MAPK signaling pathways in NSP5 phosphorylation and a negative role for GSK3β in the phosphorylation of bacterially expressed recombinant NSP5 in vitro. In the present work, using phospho-specific anti-Ser9 GSK3β antibody, we observed that GSK3β is inactivated in a rotavirus infected MA104 cells in a strain-independent manner. GSK3β¬specific small interfering RNA (siRNA-GSK3β) reduced GSK3β levels leading to increased level of synthesis of the structural rotavirus protein VP6 and NSP5 hyperphosphorylation compared to control siRNA. The pharmacological kinase inhibitors (LY294002, Genistein, PD98059, and Rapamycin) studies at the concentrations tested did not significantly affect rotavirus infection as seen from the number foci, while U0126 severely affected rotavirus replication. The results clearly demonstrated the importance of the MEK1/2 signaling pathway in the successful replication of rotavirus and NSP5 hyperphosphorylation in rotavirus-infected cells. In contrast inhibition of GSK3β activity by LiCl, increased in general, the number of foci by greater than 2-fold for all viral strains studied. These results suggest that MEK1/2 pathway majorly contributes to GSK3β inactivation in rotavirus infected cells. Thus, our results reveal that rotavirus activates both the PI3K/Akt and FAK/ERK1/2 MAPK pathways and appears to utilize them as a strategy to activate mTOR, and inhibit GSK3β through phosphorylation on serine 9, the negative regulator of rotavirus NSP5 phosphorylation, and thus facilitate translational competence of rotaviral mRNAs during virus replication cycle. It was shown previously in the laboratory by co-immunoprecipitation assay that Hsp70 interacts with rotaviral proteins VP1 and VP4 in rotavirus-infected mammalian cells. In this study, the interactions between Hsp70 with VP1 and VP4 were further evaluated in vitro by GST-pull down assay. It was observed that the N-terminal ATPase and C-terminal peptide-binding domain of Hsp70 is necessary for its direct interaction with VP1 and VP4. The presence of Hsp70 in purified double-and triple-layered virus particles further supported the observed interactions of rotaviral proteins VP1 and VP4 with Hsp70. However, the specific interaction observed between Hsp70 and rotaviral capsid proteins, VP1 and VP4 in viral particles suggests that Hsp70 has an important role during rotavirus assembly which requires further investigation.