title: Effect of the methyltransferase domain of Japanese encephalitis virus NS5 on the polymerase activity.
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 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.
authors.affiliation: Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, People's Republic of China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wang
authors.forename: Leiyun
authors.initials: L
authors.lastname: Weng
authors.forename: Xiao
authors.initials: X
authors.lastname: Tian
authors.forename: Dorian
authors.initials: D
authors.lastname: Counor
authors.forename: Jin
authors.initials: J
authors.lastname: Sun
authors.forename: Yingying
authors.initials: Y
authors.lastname: Mao
authors.forename: Vincent
authors.initials: V
authors.lastname: Deubel
authors.forename: Hidechika
authors.initials: H
authors.lastname: Okada
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Toyoda
mesh_terms: D004664:Encephalitis Virus, Japanese / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; D012324:RNA-Dependent RNA Polymerase