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Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus
S E Behrens1, L Tomei, R De Francesco
1Institut fur Virologie (FB 18), Justus-Liebig-Universitat, Giessen, Germany.
Insights
Researchers identified the Hepatitis C virus (HCV) RNA-dependent RNA polymerase, crucial for viral replication. This enzyme, non-structural protein 5B (NS5B), uses a unique
Area of Science:
- Virology
- Molecular Biology
- Hepatitis Research
Background:
- Hepatitis C virus (HCV) is a primary cause of non-A, non-B post-transfusion hepatitis.
- HCV is a (+) RNA virus with a genome encoding a large polyprotein processed into viral polypeptides.
- Viral replication involves synthesizing a complementary (-) RNA strand from the (+) RNA template.
Purpose of the Study:
- To experimentally identify the RNA-dependent RNA polymerase responsible for HCV replication.
- To characterize the enzymatic activity and mechanism of the identified HCV RNA polymerase.
Main Methods:
- Heterologous expression of viral proteins in insect cells.
- Characterization of in vitro RNA synthesis products from HCV RNA-dependent RNA polymerase.
- Purification and functional analysis of the HCV NS5B protein.
Main Results:
- Experimental evidence confirms HCV encodes an RNA-dependent RNA polymerase.
- The 65 kDa non-structural protein 5B (NS5B) possesses this enzymatic activity.
- NS5B generates dimer-sized hairpin-like RNA molecules via a 'copy-back' mechanism.
- Purified NS5B performs primer-dependent RNA synthesis on various templates.
Conclusions:
- HCV NS5B is the viral RNA-dependent RNA polymerase.
- NS5B utilizes a 'copy-back' priming mechanism for RNA polymerization.
- These findings advance the understanding of the Hepatitis C virus life cycle.
Abstract:
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.