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Semliki Forest virus replicase protein nsP2 shares sequence homology with several putative NTPases and RNA helicases. NsP2 has RNA-dependent NTPase activity. Here we expressed polyhistidine-tagged nsP2 in Escherichia coli, purified it by metal-affinity chromatography, and used it in RNA helicase assays. RNA helicase CI of plum pox potyvirus was used as a positive control. Unwinding of alpha-32P-labelled partially double-stranded RNA required nsP2, Mg2+ and NTPs. NsP2 with a mutation, K192N, in the NTP-binding sequence GVPGSGK192SA could not unwind dsRNA and had no NTPase activity. This is the first demonstration of RNA helicase activity within the large alphavirus superfamily.
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