Japanese encephalitis (JE) virus NS3 protein and two N-terminally truncated (Δ1–148 and Δ1-323) forms of NS3 were engineered and expressed in as fusion proteins with a histidine tag at the N terminus. The purified recombinant proteins his-NS3 and his-NS3 were found to possess NTPase activity which was stimulated by single-stranded RNA, whereas NS3 did not. The requirements for MgCl and MnCl and the salt and pH ranges necessary for optimal activity of the enzyme were determined and shown to be slightly different from those of the NTPases of other flaviviruses. Poly(U) and poly(C) were better than poly(A) at stimulating the NTPase activities, in contrast to other flaviviral NTPases. The substrate preference was in the order GTP > ATP ≫ UTP > CTP. Interestingly, we found that Ca could not substitute for Mg; on the contrary, it inhibited NTPase activity. The removal of the N-terminal 148 amino acids enhanced NTPase activity, but further deletion of the region (amino acids 148–323) completely abolished the activity. Therefore, amino acids 148–323 contain a critical region required for NTPase activity.


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