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.

Knowledge of the dissociation constants of the ionizable protons of weak acids in aqueous media is of fundamental importance in many areas of chemistry and biochemistry. The pKa value, or equilibrium dissociation constant, of a molecule determines the relative concentration of its protonated and deprotonated forms at a specified pH and is therefore an important descriptor of its chemical reactivity. Considerable efforts have been devoted to the determination of pKa values by different experimental techniques. Although in most cases the determination of pKa values from experimental is straightforward, there are situations where interpretation is difficult and the results ambiguous. It is, therefore, not surprising that the capability to provide accurate estimates of the pKa value has been a central goal in theoretical chemistry and there has been a large effort in developing methodologies for predicting pKa values for a variety of chemical systems by differing quantum chemical techniques. A prediction accuracy within 0.5 pKa units of experiment is the desirable level of accuracy. This is a non-trivial exercise, for an error of 1 kcal/mol in estimates of the free energy value would result in an error of 0.74 pKa units. In this thesis ab initio Car-Parrinello molecular dynamics (CPMD) has been used for investigating the Brϕnsted acid-base chemistry of weak acids in aqueous solution. A key issue in any dissociation event is how the solvating water molecules arrange themselves spatially and dynamically around the neutral and dissociated acid molecule. Ab initio methods have the advantage that all solvent water molecules can, in principle, be con- sidered explicitly. One of the factors that has inhibited the widespread use of ab initio MD methods to study the dissociation reaction is that dissociation of weak acids are rare events that require extremely long simulation times before one is observed. The metady- namics formalism provides a solution to this conundrum by preventing the system from revisiting regions of configuration space where it has been in the past. The formalism allows the system to escape the free-energy minima by biasing the dynamics with a history dependent potential (or force) that acts on select degrees of freedom, referred to as collective variables. The bias potentials, modeled by repulsive inverted Gaussians that are dropped during propagation, drive the system out of any free-energy minima and allow it to explore the configurational space by a relatively quick and efficient sampling. The the- sis deals with a detailed investigation of the Brϕnsted acid-base chemistry of weak acids in aqueous solutions by the CPMD-metadynamics procedure. In Chapter 1, current approaches for the theoretical estimation of pKa values are summarized while in Chapter 2 the simulation methodology and the metadynamics sampling techniques used in this study are described. The potential of the CPMD-metadynamics procedure to provide estimates of the acid dissociation constant (pKa) is explored in Chapter 3, using acetic acid as a test sys- tem. Using the bond-distance dependent coordination number of protons bound to the dissociating carboxylic groups as the collective variable, the free-energy profile for the dissociation reaction of acetic acid in water was computed. Convergence of the free-energy profiles and barriers for the simulations parameters is demonstrated. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid and the deterrence in their values provides the estimate for pKa. The estimated value of pKa for acetic acid from the simulations, 4.80, is in good agreement with the experiment at value of 4.76. It is shown that the good agreement with experiment is a consequence of the cancellation of errors, as the pKa values are computed as the divergence in the free energy values at the minima corresponding to the neutral and dissociated state. The chapter further explores the critical factors required for obtaining accurate estimates of the pKa values by the CPMD-metadynamics procedure. It is shown that having water molecules sufficient to complete three hydration shells as well as maintaining water density in the simulation cell as close to unity is important. In Chapter 4, the CPMD-metadynamics procedure described in Chapter-3 has been used to investigate the dissociation of a series of weak organic acids in aqueous solutions. The acids studied were chosen to highlight some of the major factors that influence the dissociation constant. These include the influence of the inductive effect, the stabilization of the dissociated anion by H-bonding as well as the presence of multiple ionizable groups. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic, the isomers of hydroxybenzoic acid and ophthalmic acids and its isomers. It was found that in each of these examples the CPMD-metadynamics procedure correctly estimates the pKa values, indicating that the formulism is capable of capturing these influences and equally importantly indicating that the cancellation of errors is indeed universal. Further, it is shown that the procedure can provide accurate estimates of the successive pKa values of polypro tic acids as well as the subtle difference in their values for different isomers of the acid molecule. Changes in protonation-deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. It is shown that CPMD simulations in conjunction with metadynamics calculations of the free energy profile of the protonation- deprotonation reaction can provide estimates of the multiple pKa values of the 20 canonical α-amino acids in aqueous solutions in good agreement with experiment (Chapter 5). The distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic and the amine groups is used as the collective variable to explore the free energy profiles of the Brϕnsted acid-base chemistry of amino acids in aqueous solutions. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule were included explicitly in the computation procedure. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error with respect to experimental results, of 0.2 pKa units. The tripeptide Glutathione (GSH) is one of the most abundant peptides and the major repository for non-protein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thioldisulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influences the redox couple and hence the pKa value of the cysteine residue of GSH is critical to its functioning. In Chapter 6, it has been reported that ab initio Car-Parrinello Molecular Dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. It is shown that the free-energy landscape for the protonation - deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared to the isolated cysteine amino acid. The dissociation constants of weak acids are commonly determined from pH-titration curves. For simple acids the determination of the pKa from the titration curves using the Henderson-Hasselbalch equation is relatively straightforward. There are situations, however, especially in polyprotic acids with closely spaced dissociation constants, where titration curves do not exhibit clear inflexion and equivalence stages and consequently the estimation of multiple pKa values from a single titration curve is no longer straightfor- ward resulting in uncertainties in the determined pKa values. In Chapter 7, the multiple dissociation constant of the hexapeptide glutathione disulfide (GSSG) with six ionizable groups and six associated dissociation constants has been investigated. The six pKa values of GSSG were estimated using the CPMD-metadynamics procedure from the free-energy profiles for each dissociation reaction computed using the appropriate collective variable. The six pKa values of GSSG were estimated and the theoretical pH-titration curve was then compared with the experimentally measured pH-titration curve and found to be in excellent agreement. The object of the exercise was to establish whether interpretation of pH-titration curves of complex molecules with multiple ionizable groups could be facilitated using results of ab initio molecular dynamics simulations.

Chapter 3 Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this chapter, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell. Further NSm was shown to be phosphorylated by N. benthamiana and tomato crude sap as observed in other movement proteins. Chapter 4 This chapter deals with localization of NSm to PD and identification of domain involved in localization. For this purpose NSm and its mutants were cloned in pEAQ:GFP vector and transiently expressed in N. benthamiana by infiltration of transformed Agrobacteria. The GFP tagged NSm was visualized by confocal microscopy. The results demonstrated that NSm forms punctate structures and localizes to PD as confirmed by colocalization of mCherry: PDLP1a, a PD marker which resides in PD, with GFP:NSm. To find out the domain involved in PD localization, sequential deletion mutants were made. It was found that C-terminal domain is involved in PD localization. On the other hand, N-terminal unfolded region was dispensable for PD localization. This is the first report of a coiled coil domain shown to be involved in PD localization. It has also been demonstrated that GBNV NSm interacts with NP. Further, membrane floatation assay carried in presence of NP suggested that interaction of NSm and NP affected membrane association of NSm. These results were further confirmed by localization studies of NSm in presence of NP. It was found that there was considerable relocalization of both NSm and NP. NSm was observed to be present in cytoplasm as well as on the membrane. At the same time, NP was observed on membrane apart from being present in the cytoplasm. When N-terminal 50 amino acids (unfolded) region of NSm was deleted and colocalization studies were carried out, it was found that NSm and NP do not colocalize, suggesting that NSm interacts with NP via the unfolded region and helps in the relocalization of NP to the membrane. Chapter 5 This chapter deals with the pathway of targeting NSm to PD. To decipher the pathway, followed by NSm, an inhibitor of endomembrane or vesicle mediated transport, Brefeldin A (BFA) was used. When GFP-NSm was expressed it was observed to form punctate structure at PD as before. Upon treatment with BFA, green islands were observed in the cytoplasm suggesting that ER was involved in targeting NSm to PD. Similarly, LatB, inhibitor of actin mediated targeting of protein to membrane, also abrogated the localization of NSm to PD. In order to further understand the role of ER in targeting NSm to PD, an ER marker, ER-GFP (GFP fused to HDEL peptide that directs it to ER) was coexpressed with GBNV NSm fused to mCherry. It was observed that NSm colocalizes with ER-GFP as yellow puncta on PD. The puncta appeared as patches and the whole ER-network was converted to vesicles. This was further confirmed by coexpressing ER-GFP with NSm without any tag. The green fluorescent vesicles were observed preferentially near cell membrane. To delineate the region of NSm involved in vesicle formation, point mutants and deletion mutants of NSm were generated without the tag and coexpressed with ER-GFP. When N-terminal 203 amino acids were deleted, NSm was able to transform ER membranes to vesicles suggesting that these residues are dispensable for vesicle formation. Interestingly, the deletion of coiled coil domain leads to cytosolic location of NSm. Furthermore, the C-terminal coiled coil domain when expressed alone was capable of inducing vesicle formation. This is the first report of involvement of such a domain in ER membrane association and vesicle formation.