An in vitro model of hepatitis C virion production

Theo Hełłer, Satoru Saito, Jonathan Auerbach, Tarice Williams, Tzivia Rachel Moreen, Allison Jazwinski, Brian Cruz, Neha Jeurkar, Ronda Sapp, Guangxiang Luo, T. Jake Liang

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2579-2583
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number7
DOIs
StatePublished - Feb 15 2005
Externally publishedYes

Fingerprint

Hepatitis C
Hepacivirus
Virion
Catalytic RNA
Viral Structural Proteins
RNA Replicase
RNA
In Vitro Techniques
Culture Media
Hepatovirus
Virus Assembly
Replicon
Mutation
Density Gradient Centrifugation
Viral RNA
Ribonucleases
Life Cycle Stages
Fluorescent Antibody Technique
Sequence Analysis
Sucrose

Keywords

  • Assembly
  • Cell culture
  • Infection
  • Ribozyme
  • Viral replication

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Hełłer, T., Saito, S., Auerbach, J., Williams, T., Moreen, T. R., Jazwinski, A., ... Liang, T. J. (2005). An in vitro model of hepatitis C virion production. Proceedings of the National Academy of Sciences of the United States of America, 102(7), 2579-2583. https://doi.org/10.1073/pnas.0409666102

An in vitro model of hepatitis C virion production. / Hełłer, Theo; Saito, Satoru; Auerbach, Jonathan; Williams, Tarice; Moreen, Tzivia Rachel; Jazwinski, Allison; Cruz, Brian; Jeurkar, Neha; Sapp, Ronda; Luo, Guangxiang; Liang, T. Jake.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 7, 15.02.2005, p. 2579-2583.

Research output: Contribution to journalArticle

Hełłer, T, Saito, S, Auerbach, J, Williams, T, Moreen, TR, Jazwinski, A, Cruz, B, Jeurkar, N, Sapp, R, Luo, G & Liang, TJ 2005, 'An in vitro model of hepatitis C virion production', Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 7, pp. 2579-2583. https://doi.org/10.1073/pnas.0409666102
Hełłer, Theo ; Saito, Satoru ; Auerbach, Jonathan ; Williams, Tarice ; Moreen, Tzivia Rachel ; Jazwinski, Allison ; Cruz, Brian ; Jeurkar, Neha ; Sapp, Ronda ; Luo, Guangxiang ; Liang, T. Jake. / An in vitro model of hepatitis C virion production. In: Proceedings of the National Academy of Sciences of the United States of America. 2005 ; Vol. 102, No. 7. pp. 2579-2583.
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