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 copyback 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/ mug/h and 2.5 muM under 0.5 mM GTP and 2.0 pmol/mug/h and 3.5 muM under 50 muM GTP, respectively. Those for copy-back RNA synthesis were similar under both conditions (k(pol) 1.8 pmol/mug/h; K-m, 3.0 muM). 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 Mg2+ to Mn2+. (C) 2002 Elsevier Science (USA).