An estimated 170 million persons worldwide are infected with hepatitis C virus (HCV), a major cause of chronic liver disease. Despite increasing knowledge of genome structure and individual viral proteins, studies on virus replication and pathogenesis have been hampered by the lack of reliable and efficient cell culture systems. A full-length consensus genome was cloned from viral RNA isolated from an infected human liver and used to construct subgenomic selectable replicons. Upon transfection into a human hepatoma cell line, these RNAs were found to replicate to high levels, permitting metabolic radiolabeling of viral RNA and proteins. This work defines the structure of HCV replicons functional in cell culture and provides the basis for a long-sought cellular system that should allow detailed molecular studies of HCV and the development of antiviral drugs.

As an initial approach to studying the molecular replication mechanisms of hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, we have recently developed selectable self-replicating RNAs. These replicons lacked the region encoding the structural proteins and instead carried the gene encoding the neomycin phosphotransferase. Although the replication levels of these RNAs within selected cells were high, the number of G418-resistant colonies was reproducibly low. In a search for the reason, we performed a detailed analysis of replicating HCV RNAs and identified several adaptive mutations enhancing the efficiency of colony formation by several orders of magnitude. Adaptive mutations were found in nearly every nonstructural protein but not in the 5 or 3 nontranslated regions. The most drastic effect was found with a single-amino-acid substitution in NS5B, increasing the number of colonies ϳ500-fold. This mutation was conserved with RNAs isolated from one cell line, in contrast to other amino acid substitutions enhancing the efficiency of colony formation to a much lesser extent. Interestingly, some combinations of these nonconserved mutations with the highly adaptive one reduced the efficiency of colony formation drastically, suggesting that some adaptive mutations are not compatible.The Hepatitis C virus (HCV) is a distinct member of the family Flaviviridae, comprising a group of enveloped viruses to which the flaviviruses, with the prototype Yellow fever virus, and the animal-pathogenic pestiviruses like Classical swine fever virus and Bovine viral diarrhea virus (BVDV) belong (41). These viruses have in common a single-stranded RNA genome of positive polarity carrying one long open reading frame (ORF) that is flanked at the 5Ј and 3Ј ends by nontranslated regions (NTRs). In HCV, the genome has a length of ϳ9,600 nucleotides and encodes a ϳ3,000-amino-acid-long polyprotein carrying the structural proteins in the amino-terminal quarter and the nonstructural (NS) proteins in the remainder (for reviews, see references 4 and 43).During and after translation, the polyprotein is cleaved in the structural region by host cell enzymes, and the NS proteins are cleaved by two viral proteinases, giving rise to at least 10 different products. These are arranged from the amino to the carboxy terminus as follows: core (C)-envelope protein 1 (E1)-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (22, 23, 54). The amino-terminal products C, E1, and E2 are the major constituents of the virus particle, and they are released from the polyprotein precursor by host cell signal peptidases (23). The function of the small hydrophobic polypeptide p7 is unknown. NS2 and the amino-terminal domain of NS3 constitute the NS2-3 proteinase, responsible for polyprotein cleavage at the NS2/3 junction (20, 24). NS3 carries two enzymatic activities residing in two well-defined globular domains (62): a chymotrypsin-like serine proteinase spanning the ϳ180 aminoterminal NS3 residues, and nucleoside triphosphatase (NTPase)/helicase activ...

The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMBO J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome. To further study the biochemical and structural properties of this enzyme, an NS5B-hexahistidine fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. The enzyme was found to have a primer-dependent RdRp activity that was able to copy a complete in vitro-transcribed HCV genome in the absence of additional viral or cellular factors. Filter binding assays and competition experiments showed that the purified enzyme binds RNA with no clear preference for HCV 3-end sequences. Binding to homopolymeric RNAs was also examined, and the following order of specificity was observed: poly(U) > poly(G) > poly(A) > poly(C). An inverse order was found for the RdRp activity, which used poly(C) most efficiently as a template but was inactive on poly(U) and poly(G), suggesting that a high binding affinity between polymerase and template interferes with processivity. By using a mutational analysis, four amino acid sequence motifs crucial for RdRp activity were identified. While most substitutions of conserved residues within these motifs severely reduced the enzymatic activities, a single substitution in motif D which enhanced the RdRp activity by about 50% was found. Deletion studies indicate that amino acid residues at the very termini, in particular the amino terminus, are important for RdRp activity but not for RNA binding. Finally, we found a terminal transferase activity associated with the purified enzyme. However, this activity was also detected with NS5B proteins with an inactive RdRp, with an NS4B protein purified in the same way, and with wild-type baculovirus, suggesting that it is not an inherent activity of NS5B.

The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome, Km values for UTP, GTP, ATP, and CTP were approximately 1.0, approximately 0.5, approximately 10, and approximately 0.3 microM, respectively. The profile of several inhibitors of RdRp activity and substrate analogs indicated that the enzyme has a strong preference for ribonucleoside 5'-triphosphates and that it closely resembles 3Dpol of picornaviruses.