Effect of the methyltransferase domain of Japanese encephalitis virus NS5 on the polymerase activity

Biochim Biophys Acta. 2012 May;1819(5):411-8. doi: 10.1016/j.bbagrm.2012.01.003. Epub 2012 Jan 21.


Japanese encephalitis virus (JEV) NS5 consists of an N-terminal guanylyltransferase/methyltransferase (MTase) domain and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. We purified JEV NS5 from bacteria and examined its RdRp activity in vitro. It showed exclusive specificity for Mn(2+) and alkaline conditions (pH 8-10) for RdRp activity. It showed strong RdRp activity with dinucleotide primers, and the order of template strength was poly(U)>(I)>(A)>(C). It showed weak transcription activity without primers, but could not transcribe poly(I) without primers. It bound homopolymeric RNA templates, but weakly bound poly(C). The Km (μM) values were 22.13±1.11 (ATP), 21.94±3.88 (CTP), 21.27±1.23 (GTP), and 9.91±0.30 (UTP), indicating low substrate affinity. Vmax (/min) values were 0.216±0.017 (ATP), 0.781±0.020 (CTP), 0.597±0.049 (GTP), and 0.347±0.022 (UTP), indicating high polymerization activity. The RdRp domain alone did not show RdRp activity; a structural and functional interaction between the MTase and RdRp domains via 299-EHPYRTWTYH-308 (MTase domain) and 739-LIGRARISPG-748 (RdRp domain) was predicted, because mutations in the MTase domain affected RdRp activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Encephalitis Virus, Japanese / enzymology*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Methyltransferases* / chemistry
  • Methyltransferases* / metabolism
  • Protein Structure, Tertiary
  • RNA-Dependent RNA Polymerase* / chemistry
  • RNA-Dependent RNA Polymerase* / metabolism
  • Viral Nonstructural Proteins* / chemistry
  • Viral Nonstructural Proteins* / metabolism


  • NS5 protein, flavivirus
  • Viral Nonstructural Proteins
  • Methyltransferases
  • RNA-Dependent RNA Polymerase