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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 a 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. An Asp-285-to-Ala substitution in motif II of the JEV NS3 protein abolished the ATPase and RNA helicase activities. These results indicate that the C-terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3.z 2000 Federation of European Biochemical Societies.

A mesophilic hydrogenotrophic methanogen, strain MHT-1 T , was isolated from the leachate of a sea-based site for solid waste disposal (the port of Osaka, Japan). Strain MHT-1 T was found to be an irregular coccus and was able to use H 2 /CO 2 and formate as energy sources. Acetate was required for growth. The optimum temperature and pH for growth were 35 mC and 6 5-7 5, respectively. Strain MHT-1 T was resistant to high concentrations of several heavy metals such as CdCl 2 and CuSO 4 . The GMC content of the DNA was 51 9 mol %. Analysis of the 16S rRNA gene revealed that the isolate was a member of the genus Methanocalculus but distinct from its nearest neighbour, Methanocalculus halotolerans, there being a sequence similarity of 98 9 %. DNA-DNA hybridization analysis revealed 51 % relatedness with the DNA of M. halotolerans strain SEBR 4845 T . The optimum NaCl concentration was 1 0%, whereas the optimum in M. halotolerans was 5 0 %. A new species, Methanocalculus pumilus, is proposed for strain MHT-1 T . The type strain is MHT-1 T (l DSM 12632 T l JCM 10627 T ).

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