Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To i nvestigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in bac ulovirus-infected insect cells and purified to near homogeneity by nickel a ffinity 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 addi tional viral and/or cellular cofactors. RdRp activity of purified NS5NHis p rotein was reduced in comparison to NS5CHis, while purified NS5NHis incorpo rating a GDD -> GVD mutation within the polymerase active site (NS5GVD) lac ked RdRp activity. RNase A digestion of the RdRp reaction products indicate d that they were double-stranded and of a similar size to the KUN replicati ve form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, simi lar to that reported for recombinant RdRp of other flaviviruses. However, i n contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demon strating efficient processivity. (C) 2001 Elsevier Science B.V. All rights reserved.