2011年1月8日，国际学术期刊Biochimica et Biophysica Acta子刊Gene regulatory mechanism在线发表了上海巴斯德研究所丰田哲也组关于肠道病毒71型(EV71) 3D聚合酶生物化学分析的研究成果。
近年来，EV71病毒引发的手足口病流行呈逐年上升的态势。自2008年截至2010年底，超过340万中国儿童感染手足口病，其中1384名患儿因严重并发症死亡。目前还没有针对EV71病毒的有效药物和疫苗。病毒RNA依赖的RNA聚合酶（RdRp）是病毒基因组编码的一类重要的酶，它催化了病毒基因组的复制和转录。由于在宿主细胞中没有类似的RNA依赖的RNA聚合酶（RdRp），所以这类聚合酶就成为了抗病毒药物开发的首选靶标。
蒋红兵博士在丰田哲也研究员的指导下，表达纯化了EV71病毒3D RNA聚合酶并对其基本生化学特性进行了鉴定。研究显示：EV71病毒RNA聚合酶是特异性依赖于Mn2+的聚合酶，在Mg2+存在条件下完全没有活性。EV71 3D聚合酶是引物依赖型的RNA聚合酶，它可以利用双核苷酸和十核苷酸RNA作为引物，以poly(C)和基因组RNA为模板从头起始转录，DNA引物如寡聚dT15可以增强其转录活性。EV71 3D聚合酶对RNA模板的结合力很弱，没有末端核苷酸转移酶活性和逆转录酶活性，其酶促反应动力学常数Km和Vmax通过经典的Lineweaver-Burk作图计算获得。EV71 3D聚合酶对GTP底物的Km值很小表明聚合酶对GTP的亲和力很高提示GTP核苷类似物可能是EV71病毒的有效抑制剂。EV71病毒的原型株BrCr-Tr和北京分离株BJ的聚合酶活性无显著性差异，而且两种不同株型的病毒聚合酶对UTP底物的动力学常数一致，这表明EV71 3D聚合酶可以作为抗病毒药物设计的靶标。
该研究得到了中国科学院和李嘉诚基金会的资助。 （生物谷Bioon.com）
生物谷推荐原文出处：
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
