Kinetic Analysis of C-Terminally Truncated RNA-Dependent RNA Polymerase of Hepatitis C Virus

• Published in: Biochemical and biophysical research communications Vol. 290; no. 4; pp. 1188 - 1194
• Main Authors: Kashiwagi, Takahito, Hara, Koyu, Kohara, Michinori, Kohara, Kyoko, Iwahashi, Jun, Hamada, Nobuyuki, Yoshino, Haruhito, Toyoda, Tetsuya
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2002
• Subjects: Animals; Base Sequence; Cell Line; DNA, Viral - genetics; Enzyme Activation - drug effects; Guanosine Triphosphate - pharmacology; Hepacivirus - enzymology; Hepacivirus - genetics; Hepatitis C virus; Kinetics; Mutation; Nucleotides - pharmacology; Peptide Fragments - genetics; Peptide Fragments - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA Replicase - genetics; RNA Replicase - metabolism; Spodoptera
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2001.6333

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/μg/h and 2.5 μM under 0.5 mM GTP and 2.0 pmol/μg/h and 3.5 μM under 50 μM GTP, respectively. Those for copy-back RNA synthesis were similar under both conditions ( k pol, 1.8 pmol/μg/h; K m, 3.0 μM). 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+.