The cis-replicating RNA elements in the 5 and 3 nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3 NTR RNA, and only poorly by 5 NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.Plus-strand RNA viruses belong to many different families, but they all have similar strategies for replicating their genomes. The nonstructural viral proteins in concert with cellular proteins form a complex with the viral RNA on membranous vesicles, where RNA replication takes place. The input RNA is first transcribed into a minus strand, which in turn serves as the template for production of progeny plus strands. For many years it was generally believed that the specificity of genome translation and replication resides in cis-acting RNA elements located in the 5Ј and 3Ј nontranslated regions (NTRs) of viral genomes. However, recently the importance of cis-replicating elements in the coding sequences of the viral polyprotein of a number of plus-strand RNA viruses has been demonstrated.Hepatitis C virus (HCV) is the major causative agent of transfusion-related and sporadic non-A, non-B hepatitis (9). HCV, a member of the Flaviviridae family, is an enveloped plus-stranded RNA virus with a genome length of about 9,600 nucleotides (nt) (reviewed in reference 52). The viral RNA contains a long 5Ј NTR, a single open reading frame, and a 3Ј NTR (Fig. 1A). The 5Ј NTR contains the IRES (internal ribosomal entry site), which promotes the translation of a large polyprotein. The virus-encoded polypeptide is processed by host and viral proteinases into at least 10 distinct proteins: NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH.Sequences of the HCV genome, the identities of viral proteins, and the basic steps in replication have been known ...