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.