title: Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin.
abstract: The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD-->GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity.
authors.affiliation: Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Australia.
authors.forename: K J
authors.initials: KJ
authors.lastname: Guyatt
authors.forename: E G
authors.initials: EG
authors.lastname: Westaway
authors.forename: A A
authors.initials: AA
authors.lastname: Khromykh
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D005416:Flavivirus / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D009154:Mutation; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D014709:Vero Cells
chemical_list: D011994:Recombinant Proteins; D012324:RNA-Dependent RNA Polymerase; D012260:Ribonucleases