The hepatitis C virus (HCV)-encoded NS5B protein is an RNA-dependent RNA polymerase which plays a substantial role in viral replication. We expressed and purified the recombinant NS5B of an HCV genotype 3a from Esherichia coli, and we investigated its ability to bind to the viral RNA and its enzymatic activity. The results presented here demonstrate that NS5B interacts strongly with the coding region of positive-strand RNA, although not in a sequence-specific manner. It was also determined that more than two molecules of polymerase bound sequentially to this region with the direction 3 to 5. Also, we attempted to determine the initiation site(s) of de novo synthesis by NS5B on X RNA, which contains the last 98 nucleotides of HCV positive-strand RNA. The initiation site(s) on X RNA was localized in the pyrimidine-rich region of stem I. However, when more than five of the nucleotides of stem I in X RNA were deleted from the 3 end, RNA synthesis initiated at another site of the specific ribonucleotide. Our study also showed that the efficiency of RNA synthesis, which was directed by X RNA, was maximized by the GC base pair at the penultimate position from the 3 end of the stem. These results will provide some clues to understanding the mechanism of HCV genomic RNA replication in terms of viral RNA-NS5B interaction and the initiation of de novo RNA synthesis.Hepatitis C virus (HCV) is the major causative agent of human viral hepatitis in most cases of non-A non-B hepatitis (5, 10). Nearly 80% of infections develop into chronic hepatitis, of which 10 to 20% progress to cirrhosis and 1 to 5% progress to hepatocellular carcinoma (22). However, neither a vaccine to prevent HCV infection nor a specific treatment for liver disease caused by chronic HCV infection is yet available. Thus, there is an urgent need to develop HCV-specific antiviral agents to counteract this human pathogen.HCV is classified as a separate genus in the family Flaviviridae (21). The virion is an enveloped virus containing a positivestrand RNA genome of 9.5 kb. The RNA genome consists of a 5Ј untranslated region (UTR), an open reading frame (ORF), and a 3Ј UTR (5,6,8). The order of the gene products of the single ORF is NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH, and this polyprotein is subsequently processed by host and viral proteases into 10 separate proteins. Among the six nonstructural proteins, the NS5B protein was recognized to be an RNA-dependent RNA polymerase (RdRp), which is the key enzyme for viral replication. Since the full-length HCV NS5B protein has poor solubility for purification, the recombinant NS5B is generally expressed on a large scale by removing the C-terminal 21 amino acid residues (28). This C-terminal region is dispensable for RdRp activity, and even more significant, this truncation positively affects its enzymatic activity (25). The HCV 3Ј UTR, which plays a major role in the initiation of RNA replication after viral infection, consists of three elements: a variable region, a poly(U-U/C) tract, and a 98-nucleotid...