An in vitro model of hepatitis C virion production
| Autor: | Theo Heller, Satoru Saito, Jonathan Auerbach, Tarice Williams, Tzivia Rachel Moreen, Allison Jazwinski, Brian Cruz, Neha Jeurkar, Ronda Sapp, Guangxiang Luo, T. Jake Liang |
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| Rok vydání: | 2005 |
| Předmět: |
Untranslated region
viruses Hepatitis C virus Hepacivirus In Vitro Techniques Biology Transfection Virus Replication medicine.disease_cause Models Biological Cell Line Viral Proteins medicine Humans RNA Catalytic Multidisciplinary Base Sequence Virion Nucleic acid sequence Ribozyme virus diseases RNA Biological Sciences Virology Molecular biology digestive system diseases NS2-3 protease Microscopy Electron Open reading frame DNA Viral Nucleic acid biology.protein Nucleic Acid Conformation RNA Viral |
| Zdroj: | Proceedings of the National Academy of Sciences. 102:2579-2583 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | The hepatitis C virus (HCV) is a major cause of liver disease worldwide. The understanding of the viral life cycle has been hampered by the lack of a satisfactory cell culture system. The development of the HCV replicon system has been a major advance, but the system does not produce virions. In this study, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5′ and 3′ ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural and nonstructural proteins were detected by immunofluorescence and Western blot. RNase protection assays showed positive- and negative-strand HCV RNA. Sequence analysis of the 5′ and 3′ ends provided further evidence of viral replication. Sucrose density gradient centrifugation of the culture medium revealed colocalization of HCV RNA and structural proteins in a fraction with the density of 1.16 g/ml, the putative density of HCV virions. Electron microscopy showed viral particles of ≈50 nm in diameter. The level of HCV RNA in the culture medium was as high as 10 million copies per milliliter. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show evidence of viral replication, assembly, and release. This system supports the production and secretion of high-level HCV virions and extends the repertoire of tools available for the study of HCV biology. |
| Databáze: | OpenAIRE |
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