Effect of the methyltransferase domain of Japanese encephalitis virus NS5 on the polymerase activity
| Autor: | Xiao Tian, Hidechika Okada, Jin Sun, Leiyun Weng, Vincent Deubel, Tetsuya Toyoda, Yingying Mao, Qiang Wang, Dorian Counor |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Guanylyltransferase
Methyltransferase GTP' viruses Biophysics Viral Nonstructural Proteins Biology Biochemistry chemistry.chemical_compound Protein structure Structural Biology Transcription (biology) RNA polymerase Genetics Molecular Biology Polymerase Encephalitis Virus Japanese RNA Methyltransferases Hydrogen-Ion Concentration RNA-Dependent RNA Polymerase Molecular biology Protein Structure Tertiary Kinetics chemistry biology.protein |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1819:411-418 |
| ISSN: | 1874-9399 |
| DOI: | 10.1016/j.bbagrm.2012.01.003 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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