RNA helicase activity of Semliki Forest virus replicase protein NSP2.
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.
Gomez de Cedrón
M
M
Centro de Biologia Molecular (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain.
Ehsani
N
N
Mikkola
M L
ML
García
J A
JA
Kääriäinen
L
L
D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012672:Semliki forest virus / Q000201:enzymology* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase; D020365:RNA Helicases