IDENTIFICATION AND PROPERTIES OF THE RNA-DEPENDENT RNA-POLYMERASE OF HEPATITIS-C VIRUS

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B post-transfusion hepatitis, Its genome, a (+)-stranded RNA molecule o f similar to 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polype ptides. As with other (+)-strand RNA viruses, the replication of HCV i s thought to proceed via the initial synthesis of a complementary (-) RNA strand, which serves, in turn, as a template for the production of progeny (+)-strand RNA molecules, An RNA-dependent RNA polymerase has been postulated to be involved in both of these steps, Using the hete rologous expression of viral proteins in insect cells, we present expe rimental evidence that an RNA-dependent RNA polymerase is encoded by H CV and that this enzymatic activity is the function of the 65 kDa non- structural protein 5B (NS5B), The characterization of the HCV RNA-depe ndent RNA polymerase product revealed that dimer-sized hairpin-like RN A molecules are generated in vitro, indicating that NS5B-mediated RNA polymerization proceeds by priming on the template via a 'copy-back' m echanism, In addition, the purified HCV NS5B protein was shown to perf orm RNA- or DNA oligonucleotide primer-dependent RNA synthesis on temp lates with a blocked 3' end or on homopolymeric templates, These resul ts represent a first important step towards a better understanding of the life cycle of the HCV.