Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn 2؉ than in the presence of Mg 2؉ . When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3 end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>50 M) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.Hepatitis C virus (HCV) is the major causative agent of non-A, non-B viral hepatitis (23). Although acute HCV infection is often asymptomatic, nearly 80% of cases resolve to chronic hepatitis, which may lead to progressive liver disease, such as cirrhosis, and liver failure. Chronic HCV infection is also associated with the development of hepatocellular carcinoma (23). It is estimated that 170 million people worldwide and more than 4 million people in the United States are currently infected with HCV (2, 65). Obviously, HCV infection remains a major threat to the public health all over the world.HCV is an enveloped RNA virus containing a singlestranded positive-sense RNA genome approximately 9.5 kb in length (14,31,56). The RNA genome consists of a 5Ј-untranslated region (5Ј UTR) of 341 nucleotides (12, 13), a large open reading frame (ORF) encoding a single polypeptide of 3,010 to 3,040 amino acids (14, 31, 56), and a 3Ј-untranslated region (3Ј UTR) of variable length (33,57,66). HCV is similar in amino acid sequence and genome organization to flaviviruses and pestiviruses (41), and therefore HCV was classified ...