西亚试剂：Biochemical characterization of enterovirus 71 3D RNA polym

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