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An in vitro Model of Hepatitis C Virion Production
Theo Heller, Satoru Saito, Jonathan Auerbach, Tarice Williams, Tzivia Rachel Moreen, Allison Jazwinski, Brian Cruz, Neha Jeurkar, Ronda Sapp, Guangxiang Luo, T. Jake Liang and Reed B. Wickner
Proceedings of the National Academy of Sciences of the United States of America
Vol. 102, No. 7 (Feb. 15, 2005), pp. 2579-2583
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/3374658
Page Count: 5
You can always find the topics here!Topics: RNA, Virions, Plasmids, Transfection, Cell lines, Viral RNA, Genotypes, Nucleotides, Replicon, Complementary DNA
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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.
Proceedings of the National Academy of Sciences of the United States of America © 2005 National Academy of Sciences