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 of approximately 9.4 kb, encodes a large polyprotein that is processed by viral and cellular proteases into at least nine different viral polypeptides. As with other (+)-strand RNA viruses, the replication of HCV is 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 heterologous expression of viral proteins in insect cells, we present experimental evidence that an RNA-dependent RNA polymerase is encoded by HCV and that this enzymatic activity is the function of the 65 kDa non-structural protein 5B (NS5B). The characterization of the HCV RNA-dependent RNA polymerase product revealed that dimer-sized hairpin-like RNA molecules are generated in vitro, indicating that NS5B-mediated RNA polymerization proceeds by priming on the template via a 'copy-back' mechanism. In addition, the purified HCV NS5B protein was shown to perform RNA- or DNA oligonucleotide primer-dependent RNA synthesis on templates with a blocked 3' end or on homopolymeric templates. These results represent a first important step towards a better understanding of the life cycle of the HCV.
Behrens
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Institut fur Virologie (FB 18), Justus-Liebig-Universitat, Giessen, Germany.
Tomei
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De Francesco
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D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D016174:Hepacivirus / Q000201:enzymology*; D008969:Molecular Sequence Data; D012313:RNA; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins; D018411:Spodoptera; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
C034477:RNA primers; D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012313:RNA; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase