Role of the DExH motif of the Japanese encephalitis virus and hepatitis C virus NS3 proteins in the ATPase and RNA helicase activities

The role of the conserved DExH motif of the Japanese encephalitis virus (JE V) NS3 protein in the ATPase and RNA helicase activities was compared with that of the hepatitis C virus (HCV) NS3 protein. In the DExH motif of JEV N S3, Asp-285 and Glu-286 were essential for both ATPase and RNA helicase act ivities. Cys-287 was critical for the RNA helicase activity of JEV NS3 but not for ATPase activity. A His-288-to-Ala substitution in the DExH motif of HCV NS3 resulted in an increase in ATPase activity which was suppressed by poly(U). In contrast, alanine substitution at the same site in JEV NS3 did not increase basal ATPase activity which remained to be stimulated by poly (U). Thus, the mutational effect at His in motif II was different in the HC V and JEV NS3 proteins. Mutagenesis at His-288 of JEV NS3 revealed that His was the most preferable amino acid for ATPase activity and Ala, Gly, Asn, Gin, 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 suggest that Cys-287 and His-288 are essential residues especially for the RNA helicase activity of JEV NS3 and the ATPase and helicase activi ties are separable enzymatic functions. (C) 2000 Academic Press.