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.

abstract: This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics. Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination. Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

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.

L’émergence récente de nouvelles thérapies antivirales efficaces est une avancée considérable pour lutter contre l'infection chronique par le virus de l'hépatite C (VHC). Cependant, un pic de carcinomes hépatocellulaires, représentant l'atteinte hépatique ultime liée à l'infection, est attendu dans la prochaine décennie. Approfondir les connaissances des différentes étapes du cycle viral et de l’interférence du VHC avec l'hépatocyte hôte permet de mieux comprendre la pathogénèse associée à ce virus. Les travaux présentés dans cette thèse ont eu pour objectif d'identifier le réseau de partenaires cellulaires et viraux de la protéine non-structurale NS2 du VHC et de mieux comprendre les mécanismes d'action et de régulation de cette protéine transmembranaire multi-fonctionnelle, qui est un acteur clé du clivage protéolytique de la polyprotéine virale et de la morphogénèse des virions. Dans une première partie, nous avons analysé comparativement les mécanismes moléculaires de l’activité enzymatique des protéines NS2 du VHC et de plusieurs hepacivirus non-humains, qui infectent des primates du Nouveau Monde (GBV-B) ou qui ont été récemment identifiés chez plusieurs autres espèces animales (NPHV, RHV, BHV et GHV). Des analyses phylogénétiques, des modèles structuraux tridimensionnels et des Études dans un contexte d'expression transitoire de précurseurs polypeptidiques viraux ou dans des modèles d'infection ont montré que l’activité des protéases NS2 de divers hepacivirus (1) s'exerce à la jonction NS2/NS3 sous la forme d'homodimères formant deux triades catalytiques composites ; (2) est régulée dans le contexte de la polyprotéine virale par quelques résidus de surface du domaine N-terminal de NS3 (NS3N) nécessaires à son activation ; (3) est efficace en l'absence complète de NS3N, suggérant un rôle négatif ou régulateur, plutôt qu'activateur de NS3N, contrairement au dogme en vigueur actuellement. Ces travaux soulignent l'importance fonctionnelle des mécanismes protéolytiques de NS2 conservés parmi les différents hepacivirus. Dans une deuxième partie, nous avons identifié un réseau de facteurs cellulaires et viraux interagissant avec NS2 au cours du cycle infectieux par un crible interactomique reposant sur la purification par affinité et l'analyse par spectrométrie de masse des complexes protéiques isolés de cellules hépatocytaires infectées, ainsi que par un test de complémentation enzymatique fonctionnelle. Par une approche d'ARN interférence, nous avons ensuite montré qu'un nombre limité de facteurs cellulaires interagissant avec NS2 sont impliqués dans la production et la sécrétion de particules virales infectieuses, incluant des protéines du complexe de la peptidase signal (SPCS) au sein du réticulum endoplasmique, des protéines chaperonnes (DNAJB11, HSPA5) et une protéine impliquée dans le transport intracellulaire (SURF4). Notamment, nos Études suggèrent que plusieurs membres du SPCS forment un complexe multi-protéique avec NS2, impliquant Également la glycoprotéine virale E2, qui jouerait un rôle dans une Étape précoce de l'assemblage ou lors de l’enveloppement de la particule virale. En conclusion, mes travaux de thèse ont permis d'identifier pour la première fois une série limitée de facteurs hépatocytaires interagissant spécifiquement avec la protéine NS2 du VHC au cours de l'infection et de déterminer parmi ceux-ci les facteurs essentiels la morphogenèse virale. Par ailleurs, nos résultats ont permis d’enrichir les connaissances naissantes des hepacivirus non-humains récemment identifiés et de montrer que ceux-ci partageaient avec le VHC des mécanismes clés mis en jeu au cours du cycle viral, ce qui contribue consolider leur intérêt comme modèles animaux de substitution. / The recent emergence of a panel of direct acting antivirals will certainly help combat chronic hepatitis C in the future. However, in the current context worldwide, a peak of hepatitis C virus (HCV)-induced hepatocellular carcinoma is expected in the next decade. Deepening our understanding of HCV life cycle and HCV interference with host cells may help monitor HCV-associated pathogenesis. The aim of my PhD work was to identify the network of host and viral interactors of HCV nonstructural protein 2 and to unravel the mechanisms of action and regulation of this multifunctional, transmembrane protein, which is key both for the viral polyprotein cleavage and virion morphogenesis.In the first part of the work, we comparatively characterized molecular mechanisms underlying the enzymatic activity of NS2 proteins from HCV and from various non-human hepaciviruses that infect small New World primates (GBV-B) or that were recently identified in the wild in several mammalian species (NPHV, RHV, BHV, GHV). A combination of phylogenetic analyses, tridimensional structural models, and studies relying on the transient expression of viral polypeptide precursors or on infection models showed that NS2 proteases of the various hepaciviruses (1) act as dimers with two composite active sites to ensure NS2/NS3 junction cleavage, (2) are regulated in the polyprotein backbone via a hydrophobic patch at the surface of NS3 N-terminal domain (NS3N) that is essential to activate NS2 protease, and (3) are efficient in the complete absence of NS3N, which is unprecedented and suggests that NS3N has rather a negative or regulating role on NS2 activity. These data underline the functional importance of NS2 proteolytic mechanisms that are conserved across hepaciviruses.In the second part, we identified a network of cellular factors and viral proteins that interact with NS2 in the course of HCV infection using an interactomic screen based on affinity purification and mass spectrometry analysis of protein complexes retrieved form HCV infected hepatoma cells, as well as a split-luciferase complementation assay. Next, using a gene silencing approach, we found that a limited set of NS2 interactors among these host factors were involved in HCV particle assembly and/or secretion. This includes members of the endoplasmic reticulum signal peptidase complex (SPCS), chaperone proteins (DNAJB11, HSPA5) and a factor involved in intracellular transport (SURF4). Notably, our data are in favor of the existence of a multiprotein complex involving NS2, several members of the SPCS, and the viral E2 glycoprotein, which likely plays a role in an early step of HCV particle assembly or during particle envelopment. Altogether, my PhD work allowed us to identify a limited set of hepatocyte factors interacting with HCV NS2 during infection and to pinpoint those that are essential for HCV morphogenesis. Additionally, our results contributed to the molecular characterization of the recently identified non-human hepaciviruses and revealed that these hepaciviruses share with HCV key mechanisms in the course of their infectious life cycles. This highlights the value of non-human hepaciviruses as surrogate animal models of HCV infection.

Eastern Equine Encephalitis virus (EEEV) causes a fatal mosquito-borne virus that is vaccine preventable for horses. The conventional serological tests measure antibodies to the structural proteins of EEEV which are also found in the vaccine. This makes it difficult to differentiate infected and vaccinated animals (DIVA). Detection of antibodies to non-structural proteins (NSPs) is a theoretical strategy that would allow you to survey natural infections among vaccinated populations. This test would also allow for more accurate representations of the natural infection rate, vaccination rate, and help identify vaccine failures. The potential uses of the NSPs of Eastern Equine Encephalitis virus as diagnostic antigens were examined in this study. Each of the four NSP encoding genes of EEEV strain FL93-939 was separated into two parts, inserted into expression vector pDEST17, and expressed in Escherichia coli strain BL21-AI. Recombinant forms of the protein were used as an antigen for an indirect IgG ELISA to measure the serological response of horse sera to the NSPs. Serum samples collected from infected, vaccinated, and unvaccinated horses were tested for NSP antibodies. A decrease in the optical densities (ODs) for the vaccinated horse sera was seen when using the NSPs compared to whole EEEV antigen. However, the ODs for the vaccinated horses were lowered to the same level as those infected, leaving no quantitative difference between the two. The use of the IgGa secondary antibody decreased the ODs even more for the vaccinated samples, but it was still impossible to differentiate the infected and vaccinated sera due to the samples' ODs being below the cutoff point. The IgGa ELISA however, was the only ELISA where the infected samples were consistently above the vaccinated samples. Based on the results of the study, it was not possible to accurately differentiate between infected and vaccinated animals. Future research should be conducted in other ways to use the NSP recombinants for the DIVA strategy. This could include the use of an IgM ELISA or microsphere immunoassay (MIA), using different IgG subtypes for the assays, using epitope mapping to develop a new recombinant protein, or the development of a DIVA vaccine.

1 Viruses are submicroscopic obligate parasites that depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. In the recent times, many new RNA viruses have evolved that possess the potential threat to plants and animals. One among them is Tospovirus (Family Bunyaviridae) which has severely affected the agricultural productivity in India. One of the Tospoviruses GBNV is a major challenge of crop production in south India. Tospoviruses shares several features such as morphology, genome structure and organization with members of other genera in the family Bunyaviridae. Virus particles are 80–120 nm in diameter. The genome includes three RNAs referred to as large (L), medium (M) and small (S). The L RNA is in negative-sense while the M and S RNAs are ambisense. The L RNA codes for the RNA-dependent RNA polymerase (RdRp), and the M RNA for the precursor of two glycoproteins (GN and GC) and a non-structural protein (NSm). The S RNA codes for the N protein and another non-structural protein (NSs). Tospovirus infection is an emerging threat for agricultural productivity in India. Therefore, biochemical and molecular characterization of these viruses is essential for developing various strategies for control of these diseases. 2 Present thesis deals with biochemical characterization of nonstructural protein, NSs of GBNV. 3 A review of literature on Tospovirus genome organization, replication, transcription, translation and assembly is presented in Chapter I. This chapter also includes the recent work on all the proteins encoded by the tospoviruses. 4 The objectives of the present study are as follows; a. Cloning, expression, purification and biophysical characterizations of rNSs. b. Analysis of its NTPase/dATPase activity c. Demonstration of nucleic acid 5’ phosphatase activity d. Characterization of nucleic acid unwinding activity of rNSs 5 The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in bacteria, protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology are described in chapter II 6 NSs of TSWV was shown to be suppressor of gene silencing (PTGS) in 2002. Since then there has been no further work on this protein. Till date neither in vitro nor in vivo study of NSs of any tospovirus has been carried out in detail. To gain insight into the biochemical function of rNSs, the NSS gene was cloned, overexpressed in E.coli and purified. The NSS gene, was cloned into pRSET-C vector. 7. Chapter 3 deals with cloning, overexpression, purification and biophysical characterization of GBNV NSs in terms of secondary structure analysis as well as its interaction with siRNA and ssRNA. The results provide the evidence that rNSs was successfully expressed in E.coli and purified (Fig. 3.1). Molecular mass of purified rNSs was confirmed by MALDI TOF, which gave the molecular mass of expected size 51.5 kDa (Fig. 3.2) Circular dichroism study revealed that rNSs has negative ellipticity peak at 215 and 223 nm typical of a globular protein. The protein had an emission maximum at 340 nm (Fig 3.3 B) when exited at 280 nm, which reflects that rNSs is well folded. Thermal melting study (Fig 3.3 C) showed rNSs had a reasonably high Tm (65°C). So overall, spectral study suggested that purified rNSs was soluble, well folded and thermally stable and could be used for further biochemical assay. The oligomeric status of the protein was determined by size exclusion chromatography to be trimeric (156 kDa, Fig 3.5). Purified rNSs was used to raise the polyclonal antibodies in rabbit. The antiserum could detect rNSs specific band only in IPTG induced sample not in uninduced sample (Fig 3.6). 50% binding was observed at 100 ng/ml of antigen showing that these antibodies were of high affinity (Fig 3.7 B). Further, the 50% binding was observed at 1:34000 dilution of the antiserum, which suggests that high titer antibodies against rNSs were obtained (Fig 3.7 A). 8 Further, the RNA binding property of rNSs was examined. Synthetic 21 bp siRNA and in vitro transcribed 100 nt ssRNA was used to analyze the RNA binding property of rNSs. Indeed rNSs was able to bind with 100 nt ssRNA (Fig 3.8 A) or 21 nt siRNA in a protein concentration dependent manner (Fig 3.8 B). The binding however did not require presence of divalent cation such as Mg 2+ (Fig 3.8 C). In order to understand the biological function of rNSs, its interaction with the structural protein, NP by ELISA was investigated. rNSs could interact with the NP protein (Fig 3.9) . Further 15 amino deletions from C terminus of NP did not affect its interaction with rNSs protein (Fig 3.9), which suggest that the C terminal 15 amino acid residues of NP are not essential for interaction with rNSs in vitro. 9. Sequence analysis of GBNV NSs revealed the presence of Walker motifs A (GxxxxGKT) and B (DExx) in its primary structure (Fig 4.2). The proteins that possess the Walker motifs A and B exhibit ATPase activity. Therefore, the purified rNSs was tested for its ability to hydrolyze ATP in the absence and presence of poly(A) (chapter IV). rNSs could hydrolyze [γ-32P] ATP in a concentration-dependent manner (Fig. 4.3 A). Further, ATPase activity was stimulated in presence of poly(A) (Fig. 4.3 B). Quantitative analysis of reaction product suggested that the reaction was linear in the presence of poly(A) upto 1.6 µg of rNSs (Fig. 4.3 C). 10. The product of ATP hydrolysis by rNSs had the same mobility as the phosphate released by RecoP51 ATPase, a positive control used in the assay. In contrast, another viral protein from the Cotton leaf curl virus, His tagged-AV2, purified in same way as rNSs, did not show the release of phosphate, suggesting that the activity was not due to the histidine tag present at the N-terminus of rNSs. Further, no release of phosphate could be seen when immunodepleted rNSs was used suggesting that the activity was inherent to the protein and was not due to bacterial contamination (Fig 4.3 lane 7). Time course analysis of ATPase activity revealed that the reaction is linear up to 25 mins (Fig 4.4). Further, pH profile was a typical bell shaped curve with a distinct pH optimum at pH 7.0 (Fig 4.5 A) and the temperature optimum was at 25 °C(Fig 4.5 B). Most of the known viral ATPases require the divalent cation for their activity. The rNSs exhibited the optimum ATPase activity between 2-2.5 mM of MgCl2. The reaction was inhibited by increasing concentration of EDTA demonstrating the requirement of Mg2+ for ATP hydrolysis (Fig. 4.7). Further, the ATPase activity of rNSs was inhibited by increasing concentrations of non-hydrolyzable analog of ATP (Fig. 4.8) and was not inhibited by AMP (Fig 4.9) suggesting that rNSs is not a nucleotidyl phosphatase and is a true ATPase. Limited proteolysis of rNSs suggested that core domain was 23 kDa in size and could catalyze ATP hydrolysis (Fig. 21 and 4.22). 11. Interestingly rNSs not only cleaved ATP rather it could hydrolyze all rNTPs as well as dATP (Fig 4.10). Kinetic parameters were determined for its enzymatic activity. Comparison of the kinetic constants of rNSs NTPase activity revealed little variation, suggesting that the rNSs has a broad substrate specificity (Fig 4.10- 4.15 and table 4.1). 12. To assess the role of amino acids in Walker motif A and B (Fig. 4.16) site specific mutants K189A and D159A were generated ( Fig 4.17) confirmed by sequencing, overexpressed in E.coli and purified (Fig. 4.18). Point mutation in Walker motif B (D159A) reduced the ATPase activity (Fig 4.19) where as point mutation in Walker motif A (K189A abolishes the activity (Fig 4.19). 13. Chapter V deals with the nucleic acid 5’ phosphatase activity of rNSs. Experimental evidence presented in this chapter clearly shows that rNSs can cleave the single phosphate from the ssDNA, ssRNA, dsRNA and dsDNA. Nucleic acid 5’ phosphatase activity of rNSs was inhibited by AMP and ATP (Fig 5.2 and Fig 5.3). Interestingly the K189A mutant rNSs was as active as wild type rNSs where as D159A mutant showed slightly reduced activity (Fig 5.7 C). 14. As mentioned earlier, rNSs was shown to possesses the RNA stimulated NTPase/dATPase activity, a hallmark of all known helicases. Therefore, its nucleic acid unwinding activity was examined using dsDNA and dsRNA as a substrate. rNSs was able to unwind the dsDNA as well as dsRNA in a ATP dependent manner (chapter VI, Fig. 6.1 and 6.5 respectively). ATP and Mg2+ are essential cofactors for the unwinding activity (Fig. 6.1). While the unwinding activity could be observed with ATP and to some extent with dATP, all other NTPs and dNTPs failed to support the helicase function of rNSs (Fig 6.2) Further experimental evidence suggested that rNSs is a bidirectional helicase (Fig. 6.3). D159A mutation in Walker motif B resulted in reduced helicase activity where as K189A mutation in walker Motif A completely abolished the DNA as well as RNA helicase activity of rNSs (Fig. 6.6 and Fig 6.7 respectively). Therefore, mutational analysis clearly suggests that helicase activity is an intrinsic property of rNSs. 15. In conclusion rNSs of GBNV is multifunctional enzyme. This is the first report on the demonstration that rNSs is an non canonical ATP dependent helicase in the Bunyaviridae family. In addition to being a suppressor of PTGS, NSs may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. This new research finding on NSs might pave way for further studies on its role in viral replication and transcription.

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No description available.

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.

Rubella virus (RUBV) is a positive-strand RNA virus and the causative agent of rubella and congenital rubella syndrome in humans. To replicate its RNA, RUBV forms membrane-associated spherules, called replication complexes (RCs), the induction of which requires the two virus nonstructural proteins (NSPs), P150 and P90. Interestingly, late in infection the NSPs form a unique cytoplasmic fiber network, similar in appearance to microtubules, the function of which is unknown. Little is known about the roles of the RUBV NSPs in forming these structures and, to this end, we scrutinized the behavior and biochemical properties of the NSPs, both after expression from plasmids and during RUBV infection, using mutagenic, biochemical and pharmacological approaches. The following findings were made: First, the precursor from which P150 and P90 are produced via an embedded protease at the C-terminus of P150, called P200, was required for initial targeting to cytoplasmic foci. P150 was the determinant of fiber formation and while P90 had no specific targeting sequences on its own, P90 sequences within P200 were required for correct targeting of P200. An alpha-helix at the N-terminus of P150 was also important for correct targeting of P200, putatively by mediating the interaction between P150 and P90 within the precursor. Second, the membrane binding domain within the NSPs was within the N-terminal ~450 amino acids of P150. P150 is in an exceptionally tight association with membranes. Third, both the N- and C-terminal regions of P150, and specifically long alpha-helices within these regions, are necessary for fiber formation. Fiber formation relied on an intact microtubule network, but neither microtubule repositioning nor dynamic movement along microtubules was required. Additionally, it was shown that microtubules were not necessary in RUBV replication. Finally, P150 fibers were not required for RUBV replication; however, it was shown that the fibers are likely important in formation of cytoplasmic extensions through which a novel system of cell-to-cell transport of viral RNA in the absence of virus particles appears to occur.