Study on the RNA Helicase Activity of the Flavivirus NS3 Proteins

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The NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA Helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with His-tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP This result indicate that the C-terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3. Several amino acid substitutions were introduced at the DExH motif of JEV NS3, Asp-285 and Glue-286 were found essential for both ATPase and RNA helicase activities. Cys-287 was critical for the RNA helicase activity of JEV NS3 but not for ATPase activity. Mutagenesis at His-288 of JEV NS3 revealed that His was the most preferable amino acid for ATPase activity and Ala, Gly, Asn, Gln, Ser, or Arg could partly substitute for it. However, any other mutation at His-288 completely disrupted the RNA helicase activity of JEV NS3. The results suggested that Cys-287 and His-288 are essential residues especially for the RNA helicase activity of JEV NS3 and ATPase and helicase activities are separable enzymatic functions.