ATPase activity was assayed as described in the methods section using [c-32 P] ATP as the substrate. The radiolabelled Pi released was monitored by TLC on PEI cellulose plates. As mentioned earlier, proteins which posses the Walker A and Walker B motifs are known to exhibit the RNA/DNA stimulated NTPase activity [15] . Therefore, purified rNSs (0.4-2 mg) was used to determine the ATPase activity in the absence and presence of Poly (A). As shown in Fig. 3 , rNSs could hydrolyze [c-32 P] ATP in a concentrationdependent manner. The activity was higher in the presence of poly(A) (Fig. 3B ). The product of ATP hydrolysis by rNSs had the same mobility as the phosphate released by RecoP51 ATPase which was used as the positive control ( Fig. 3A and B, lane 6). In contrast, another viral protein from the Cotton leaf curl virus, His tagged-AV2, did not show the release of phosphate ( Fig. 3A and B, lane 8), suggesting that the activity was not due to the histidine tag present at the N-terminus of rNSs. Further, no release of phosphate was detected following immunodepletion of rNSs, confirming that the activity was inherent to the NSs protein ( Fig. 3A and B, lane 7). The reaction was completely inhibited by 10 mM EDTA ( Fig. 3A and B, lane 9) suggesting that the reaction is metal-ion dependent. The amount of product (phosphate) released was quantitated as described in the methods section. There was a 10-fold increase in the activity in the presence of poly(A) as compared to that without poly(A) (Fig. 3C) . The reaction was linear in the presence of poly(A) upto 1.6 mg of rNSs (Fig. 3C ). Since the ATPase activity of rNSs was much higher in the presence of poly(A), all further standardization of reaction conditions was carried out in the presence of poly(A). The product release was linear up to 30 mins (Fig. S2A ) and the ATPase activity of rNSs was enhanced by the addition of MgCl 2 . Maximum activity was observed at 2.5 mM of MgCl 2 (Fig. S2B) . Addition of increasing concentrations of EDTA lead to inhibition of activity and the ATP cleavage was completely inhibited at 5 mM EDTA (Fig. S2C) . The pH profile was a typical bell shaped curve with a distinct pH optimum at pH 7.0 (Fig. S2D ) and the optimum temperature of the reaction was 25uC (data not shown).