1. Kinetic Analysis of C-Terminally Truncated RNA-Dependent RNA Polymerase of Hepatitis C Virus
- Author
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Nobuyuki Hamada, Haruhito Yoshino, Tetsuya Toyoda, Koyu Hara, Michinori Kohara, Takahito Kashiwagi, Jun Iwahashi, and Kyoko Kohara
- Subjects
GTP' ,Hepatitis C virus ,Biophysics ,RNA-dependent RNA polymerase ,Hepacivirus ,Spodoptera ,Guanosine triphosphate ,Biology ,medicine.disease_cause ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,RNA polymerase ,medicine ,Animals ,Transferase ,Nucleotide ,Molecular Biology ,chemistry.chemical_classification ,Base Sequence ,Nucleotides ,Cell Biology ,Ribosomal RNA ,RNA-Dependent RNA Polymerase ,Molecular biology ,Peptide Fragments ,Recombinant Proteins ,Enzyme Activation ,Kinetics ,chemistry ,DNA, Viral ,Mutation ,Guanosine Triphosphate - Abstract
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+).
- Published
- 2002
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