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

J Virol Methods. 2001 Mar;92(1):37-44. doi: 10.1016/s0166-0934(00)00270-6.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Binding Sites
  • Chlorocebus aethiops
  • Chromatography, Affinity
  • Flavivirus / enzymology
  • Flavivirus / genetics*
  • Genetic Vectors*
  • Mutation
  • RNA Replicase / genetics*
  • RNA Replicase / isolation & purification
  • RNA Replicase / metabolism
  • Recombinant Proteins / metabolism
  • Ribonucleases / metabolism
  • Vero Cells

Substances

  • Recombinant Proteins
  • RNA Replicase
  • Ribonucleases