Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon Free

Abstract

Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing -transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

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2005-11-01
2024-03-28
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References

  1. Blight K. J., Kolykhalov A. A., Rice C. M. 2000; Efficient initiation of HCV RNA replication in cell culture. Science 290:1972–1974 [CrossRef]
    [Google Scholar]
  2. Bukh J., Pietschmann T., Lohmann V. 7 other authors 2002; Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc Natl Acad Sci U S A 99:14416–14421 [CrossRef]
    [Google Scholar]
  3. Date T., Kato T., Miyamoto M., Zhao Z., Yasui K., Mizokami M., Wakita T. 2004; Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells. J Biol Chem 279:22371–22376 [CrossRef]
    [Google Scholar]
  4. Egger D., Wolk B., Gosert R., Bianchi L., Blum H. E., Moradpour D., Bienz K. 2002; Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76:5974–5984 [CrossRef]
    [Google Scholar]
  5. Frese M., Pietschmann T., Moradpour D., Haller O., Bartenschlager R. 2001; Interferon- α inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway. J Gen Virol 82:723–733
    [Google Scholar]
  6. Friebe P., Lohmann V., Krieger N., Bartenschlager R. 2001; Sequences in the 5′ nontranslated region of hepatitis C virus required for RNA replication. J Virol 75:12047–12057 [CrossRef]
    [Google Scholar]
  7. Gosert R., Egger D., Lohmann V., Bartenschlager R., Blum H. E., Bienz K., Moradpour D. 2003; Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77:5487–5492 [CrossRef]
    [Google Scholar]
  8. Guo J. T., Bichko V. V., Seeger C. 2001; Effect of alpha interferon on the hepatitis C virus replicon. J Virol 75:8516–8523 [CrossRef]
    [Google Scholar]
  9. Huang L., Sineva E. V., Hargitti M. R. S., Sharma S. D., Suthar M., Raney K. D., Cameron C. E. 2004; Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli . Protein Expr Purif 37:144–153 [CrossRef]
    [Google Scholar]
  10. Kato T., Furusaka A., Miyamoto M., Date T., Yasui K., Hiramoto J., Nagayama K., Tanaka T., Wakita T. 2001; Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol 64:334–339 [CrossRef]
    [Google Scholar]
  11. Kato T., Date T., Miyamoto M., Furusaka A., Tokushige K., Mizokami M., Wakita T. 2003; Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125:1808–1817 [CrossRef]
    [Google Scholar]
  12. Kato T., Date T., Miyamoto M., Zhao Z., Mizokami M., Wakita T. 2005; Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon. J Virol 79:592–596 [CrossRef]
    [Google Scholar]
  13. Krieger N., Lohmann V., Bartenschlager B. 2001; Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75:4614–4624 [CrossRef]
    [Google Scholar]
  14. Lohmann V., Korner F., Koch J. O., Herian U., Theilmann L., Bartenschlager R. 1999; Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110–113 [CrossRef]
    [Google Scholar]
  15. Lohmann V., Hoffmann S., Herian U., Penin F., Bartenschlager R. 2003; Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol 77:3007–3019 [CrossRef]
    [Google Scholar]
  16. Moradpour D., Kary P., Rice C. M., Blum H. E. 1998; Continuous human cell lines inducibly expressing hepatitis C virus structural and nonstructural proteins. Hepatology 28:192–201
    [Google Scholar]
  17. Wakita T., Pietschmann T., Kato T. 9 other authors 2005; Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796 [CrossRef]
    [Google Scholar]
  18. Wang C., Pflugheber J., Sumpter R. Jr, Sodora D. L., Hui D., Sen G. C., Gale M. Jr 2003; Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication. J Virol 77:3898–3912 [CrossRef]
    [Google Scholar]
  19. Yi M., Bodola F., Lemon S. M. 2002; Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein. Virology 304:197–210 [CrossRef]
    [Google Scholar]
  20. Zhong J., Gastaminza P., Cheng G. 7 other authors 2005; Robust hepatitis C virus infection in vitro . Proc Natl Acad Sci U S A 102:9294–9299 [CrossRef]
    [Google Scholar]
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