title: Biochemical characterization of enterovirus 71 3D RNA polymerase.
abstract: An unusual enterovirus 71 (EV71) epidemic has begun in China since 2008. EV71 RNA polymerases (3D(pol)) showed polymerase activity with an Mn(2+). Little activity was detected with Co(2+), and no activity was detected with Mg(2+), Ca(2+), Cu(2+), Ni(2+), Cd(2+), or Zn(2+). 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 3D(pol). However, EV71 3D(pol) 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 3D(pol) 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 3D(pol) 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 3D(pol) as an antiviral drug target.
authors.affiliation: Unit of Viral Genome Regulation, Institut Pasteur of Shanghai, Key Laboratory of Molecular Virology & Immunology, Chinese Academy of Sciences, Shanghai, PR China.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Jiang
authors.forename: Leiyun
authors.initials: L
authors.lastname: Weng
authors.forename: Na
authors.initials: N
authors.lastname: Zhang
authors.forename: Minetaro
authors.initials: M
authors.lastname: Arita
authors.forename: Renqing
authors.initials: R
authors.lastname: Li
authors.forename: Lijuan
authors.initials: L
authors.lastname: Chen
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Toyoda
mesh_terms: D045744:Cell Line, Tumor; D017931:DNA Primers / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D029821:Enterovirus A, Human / Q000201:enzymology* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases