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

Qiang Wang, Leiyun Weng, Xiao Tian, Dorian Counor, Jin Sun, Yingying Mao, Vincent Deubel, Hidechika Okada, Tetsuya Toyoda

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

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 Mn2+ 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.

Original languageEnglish (US)
Pages (from-to)411-418
Number of pages8
JournalBiochimica et Biophysica Acta - Gene Regulatory Mechanisms
Volume1819
Issue number5
DOIs
StatePublished - May 2012

Keywords

  • Japanese encephalitis virus
  • Km
  • Methyltransferase
  • NS5
  • RNA polymerase
  • Vmax

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics

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