2011年1月8日,，國際學(xué)術(shù)期刊Biochimica et Biophysica Acta子刊Gene regulatory mechanism在線發(fā)表了上海巴斯德研究所豐田哲也組關(guān)于腸道病毒71型(EV71) 3D聚合酶生物化學(xué)分析的研究成果,。
近年來，EV71病毒引發(fā)的手足口病流行呈逐年上升的態(tài)勢,。自2008年截至2010年底,，超過340萬中國兒童感染手足口病，其中1384名患兒因嚴(yán)重并發(fā)癥死亡,。目前還沒有針對EV71病毒的有效藥物和疫苗,。病毒RNA依賴的RNA聚合酶（RdRp）是病毒基因組編碼的一類重要的酶，它催化了病毒基因組的復(fù)制和轉(zhuǎn)錄,。由于在宿主細(xì)胞中沒有類似的RNA依賴的RNA聚合酶（RdRp）,，所以這類聚合酶就成為了抗病毒藥物開發(fā)的首選靶標(biāo)。
蔣紅兵博士在豐田哲也研究員的指導(dǎo)下,，表達(dá)純化了EV71病毒3D RNA聚合酶并對其基本生化學(xué)特性進(jìn)行了鑒定,。研究顯示：EV71病毒RNA聚合酶是特異性依賴于Mn2+的聚合酶，在Mg2+存在條件下完全沒有活性,。EV71 3D聚合酶是引物依賴型的RNA聚合酶,，它可以利用雙核苷酸和十核苷酸RNA作為引物，以poly(C)和基因組RNA為模板從頭起始轉(zhuǎn)錄,，DNA引物如寡聚dT15可以增強(qiáng)其轉(zhuǎn)錄活性,。EV71 3D聚合酶對RNA模板的結(jié)合力很弱，沒有末端核苷酸轉(zhuǎn)移酶活性和逆轉(zhuǎn)錄酶活性,，其酶促反應(yīng)動力學(xué)常數(shù)Km和Vmax通過經(jīng)典的Lineweaver-Burk作圖計(jì)算獲得,。EV71 3D聚合酶對GTP底物的Km值很小表明聚合酶對GTP的親和力很高提示GTP核苷類似物可能是EV71病毒的有效抑制劑。EV71病毒的原型株BrCr-Tr和北京分離株BJ的聚合酶活性無顯著性差異,，而且兩種不同株型的病毒聚合酶對UTP底物的動力學(xué)常數(shù)一致,，這表明EV71 3D聚合酶可以作為抗病毒藥物設(shè)計(jì)的靶標(biāo)。
該研究得到了中國科學(xué)院和李嘉誠基金會的資助,。 （生物谷Bioon.com）
生物谷推薦原文出處：
Biochimica et Biophysica Acta doi:10.1016/j.bbagrm.2011.01.001
Biochemical characterization of enterovirus 71 3D RNA polymerase
Hongbing Jianga, Leiyun Wenga, Na Zhanga, Minetaro Aritab, Renqing Lic, Lijuan Chenc and Tetsuya Toyoda
Abstract
An unusual enterovirus 71 (EV71) epidemic has begun in China since 2008. EV71 RNA polymerases (3Dpol) showed polymerase activity with an Mn2+. Little activity was detected with Co2+, and no activity was detected with Mg2+, Ca2+, Cu2+, Ni2+, Cd2+, or Zn2+. It is a primer-dependent polymerase, and the enzyme functioned with both di- and 10-nucleotide RNA primers. DNA primer, dT15, increased primer activity, similar to other enterovirus 3Dpol. However, EV71 3Dpol initiated de novo transcription with a poly(C) template and genome RNA. Its RNA binding activity was weak. Terminal nucleotidyl transferase and reverse transcriptase activity were not detected. The Km and Vmax for EV71 3Dpol were calculated from classic Lineweaver–Burk plots. The Km values were 2.35 ± 0.05 (ATP), 5.40 ± 0.93 (CTP), 1.12 ± 0.10 (GTP) and 2.81 ± 0.31 (UTP), and the Vmax values were 0.00078 ± 0.00005/min (ATP), 0.011 ± 0.0017/min (CTP), 0.050 ± 0.0043/min (GTP) and 0.0027 ± 0.0005/min (UTP). The Km of EV71 3Dpol was similar to that of foot and mouth disease virus and rhinovirus. Polymerase activity of BrCr-TR strain and a strain from a clinical isolate in Beijing, 2008 were similar, indicating the potential for 3Dpol as an antiviral drug target.
Research Highlights
We analyzed biochemical character of enterovirus (EV) 71 3D RNA polymerase (3Dpol). ? Since 2008 China has suffered from unusual EV71 epidemic. 3Dpol of the prototype strain and the Chinese isolate showed the similar activity. ? Our EV71 3Dpol showed polymerase activity only with Mn2+. ?Our EV71 3Dpol showed low template RNA binding activity.
Keywords: Enterovirus 71; 3D protein; RNA polymerase; Transcription; Km; Vmax
Abbreviations: 3Dpol, 3D polymerase; BJ, Beijing; cre, cis-acting replication element; CV, coxsackie virus; E. coli, Escherichia coli; EV71, enterovirus 71; FBS, fetal bovine sera; FMDV, foot-and-mouth disease virus; HCV, hepatitis C virus; HFMD, hand foot mouth disease; HRV, human rhino virus; IRES, internal ribosome entry site; MEM, minimal essential media; MuLV, murine leukemia virus; PAGE, polyacrylamide gel electrophoresis; RD, rhabdomyosarcoma; RTase, reverse transcriptase; RdRp, RNA dependent RNA polymerase; SPR, surface plasmon resonance; Tm, melting temperature; knt, kilo-nucleotide; nt, nucleotide
