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

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 (mu M) 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. (C) 2012 Elsevier B.V. All rights reserved.