CHARACTERIZATION OF THE NTPASE ACTIVITY OF JAPANESE ENCEPHALITIS-VIRUS NS3 PROTEIN

Japanese encephalitis (JE) virus NS3 protein and two N-terminally trun cated (Delta 1-148 and Delta 1-323) forms of NS3 were engineered and e xpressed in E. coli as fusion proteins with a histidine tag at the N t erminus. The purified recombinant proteins his-NS3 and his-NS3((Delta 1-148)) were found to possess NTPase activity which was stimulated by single-stranded RNA, whereas NS3((Delta 1-323)) did not, The requireme nts for MgCl2 and MnCl2 and the salt and pH ranges necessary for optim al activity of the enzyme were determined and shown to be slightly dif ferent from those of the NTPases of other flaviviruses, Poly(U) and po ly(C) were better than poly(A) at stimulating the NTPase activities, i n contrast to other flaviviral NTPases, The substrate preference was i n the order GTP > ATP much greater than UTP > CTP, Interestingly, we f ound that Ca2+ could not substitute for Mg2+; on the contrary, it inhi bited NTPase activity, The removal of the N-terminal 148 amino acids e nhanced NTPase activity, but further deletion of the region (amino aci ds 148-323) completely abolished the activity, Therefore, amino acids 148-323 contain a critical region required for NTPase activity.