Kinetic analysis of C-terminally truncated RNA-dependent RNA polymerase of hepatitis C virus.
The biochemical properties of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) truncated with C-terminal 21 amino acids and expressed in insect cells were analyzed. The enzyme carried copy-back and de novo RNA synthesis activity but not terminal nucleotidyl transferase activity. k(pol) and K(m) for de novo RNA synthesis were calculated as 10.0 pmol/microg/h and 2.5 microM under 0.5 mM GTP and 2.0 pmol/microg/h and 3.5 microM under 50 microM GTP, respectively. Those for copy-back RNA synthesis were similar under both conditions (k(pol), 1.8 pmol/microg/h; K(m), 3.0 microM). De novo RNA synthesis was activated by 0.5 mM GTP. However, the ratio of GTP to three other NTPs was important for activation. Our HCV RdRp showed high activity for the complementary sequence of the HCV internal ribosomal entry site and a synergistic effect of Mg(2+) to Mn(2+).
Kashiwagi
Takahito
T
Department of Virology, Kurume University, School of Medicine, 67 Asahimachi, Kurume, Fukuoka, 830-0011, Japan.
Hara
Koyu
K
Kohara
Michinori
M
Kohara
Kyoko
K
Iwahashi
Jun
J
Hamada
Nobuyuki
N
Yoshino
Haruhito
H
Toyoda
Tetsuya
T
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D009711:Nucleotides / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D004279:DNA, Viral; D009711:Nucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D012324:RNA-Dependent RNA Polymerase