Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase
| Autor: | Radhakrishnan Rathnachalam, Joseph M. Colacino, Karla Kirkegaard, Q. May Wang, Robert B. Johnson, Kirk A. Staschke, Steve Parsons, Katharine A. Case, Jirong Lu, Michelle A. Hockman, Faming Zhang |
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| Rok vydání: | 2002 |
| Předmět: |
Hot Temperature
Hepatitis C virus viruses Immunology RNA-dependent RNA polymerase Replication Hepacivirus Viral Nonstructural Proteins medicine.disease_cause Microbiology chemistry.chemical_compound Transcription (biology) Virology RNA polymerase Two-Hybrid System Techniques Enzyme Stability medicine RNA polymerase I NS5B Polymerase biology virus diseases biochemical phenomena metabolism and nutrition RNA-Dependent RNA Polymerase Molecular biology digestive system diseases NS2-3 protease Cross-Linking Reagents chemistry Biochemistry Insect Science biology.protein RNA Viral Crystallization |
| Zdroj: | Journal of virology. 76(8) |
| ISSN: | 0022-538X |
| Popis: | The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K d , of ≈22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data. |
| Databáze: | OpenAIRE |
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