日本脑炎病毒NS5甲基转移酶结构域对聚合酶活性的影响。
Effect of the methyltransferase domain of Japanese encephalitis virus NS5 on the polymerase activity.
作者信息
Wang Qiang, Weng Leiyun, Tian Xiao, Counor Dorian, Sun Jin, Mao Yingying, Deubel Vincent, Okada Hidechika, Toyoda Tetsuya
机构信息
Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, People's Republic of China.
出版信息
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.
日本脑炎病毒（JEV）的NS5由一个N端鸟苷酸转移酶/甲基转移酶（MTase）结构域和一个C端RNA依赖性RNA聚合酶（RdRp）结构域组成。我们从细菌中纯化了JEV NS5，并在体外检测了其RdRp活性。它对Mn(2+)具有专一性，且RdRp活性的适宜碱性条件为pH 8 - 10。它对二核苷酸引物表现出很强的RdRp活性，模板强度顺序为聚（U）>（I）>（A）>（C）。在没有引物的情况下它表现出较弱的转录活性，但在没有引物时无法转录聚（I）。它能结合同聚物RNA模板，但与聚（C）的结合较弱。Km（μM）值分别为22.13±1.11（ATP）、21.94±3.88（CTP）、21.27±1.23（GTP）和9.91±0.30（UTP），表明底物亲和力较低。Vmax（/min）值分别为0.216±0.017（ATP）、0.781±0.020（CTP）、0.597±0.049（GTP）和0.347±0.022（UTP），表明聚合活性较高。单独的RdRp结构域不显示RdRp活性；通过299 - EHPYRTWTYH - 308（MTase结构域）和739 - LIGRARISPG - 748（RdRp结构域）预测MTase和RdRp结构域之间存在结构和功能相互作用，因为MTase结构域中的突变会影响RdRp活性。
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