1887

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Volume 85, Issue 7

Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon Free

Abstract

An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture. It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B. However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins. The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons. This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture. With the use of a -lactamase reporter gene as a marker for HCV replication and Man RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated. Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons. This feature was characterized as a dominant negative effect. Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon. In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons.

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2004-07-01
2024-04-23
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References

  1. Bartenschlager R., Lohmann V. 2001; Novel cell culture systems for the hepatitis C virus. Antiviral Res 52:1–17 [CrossRef]
     [Google Scholar]
  2. 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]
  3. Blight K. J., McKeating J. A., Rice C. M. 2002; Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76:13001–13014 [CrossRef]
     [Google Scholar]
  4. Blight K. J., McKeating J. A., Marcotrigiano J., Rice C. M. 2003; Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. J Virol 77:3181–3190 [CrossRef]
     [Google Scholar]
  5. 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]
  6. Chung K. M., Lee J., Kim J., Song O., Cho S., Lim J., Seedorf M., Hahm B., Jang S. K. 2000; Nonstructural protein 5A of hepatitis C virus inhibits the function of karyopherin beta3. J Virol 74:5233–5241 [CrossRef]
     [Google Scholar]
  7. De Francesco R., Rice C. M. 2003; New therapies on the horizon for hepatitis C: are we close?. Clin Liver Dis 7:211–242 [CrossRef]
     [Google Scholar]
  8. Foster G. R., Thomas H. C. 2000; Therapeutic options for HCV – management of the infected individual. Baillières Best Pract Res Clin Gastroenterol 14:255–264 [CrossRef]
     [Google Scholar]
  9. 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]
  10. Ghosh A. K., Majumder M., Steele R., Yaciuk P., Chrivia J., Ray R., Ray R. 2000; Hepatitis C virus NS5A protein modulates transcription through a novel cellular transcription factor SRCAP. J Biol Chem 275:7184–7188 [CrossRef]
     [Google Scholar]
  11. Grassmann C. W., Isken O., Tautz N., Behrens S.-E. 2001; Genetic analysis of the pestivirus nonstructural coding region: defects in the NS5A unit can be complemented in trans . J Virol 75:7791–7802 [CrossRef]
     [Google Scholar]
  12. Grobler J. A., Markel E. J., Fay J. F., Graham D. J., Simcoe A. L., Ludmerer S. W., Murray E. M., Migliaccio G., Flores O. A. 2003; Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase. J Biol Chem 278:16741–16746 [CrossRef]
     [Google Scholar]
  13. 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]
  14. He Y., Nakao H., Tan S., Polyak S. J., Neddermann P., Vijaysri S., Jacobs B. L., Katze M. G. 2002; Subversion of cell signaling pathways by hepatitis C virus nonstructural 5A protein via interaction with Grb2 and P85 phosphatidylinositol 3-kinase. J Virol 76:9207–9217 [CrossRef]
     [Google Scholar]
  15. Ikeda M., Yi M., Li K., Lemon S. M. 2002; Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 76:2997–3006 [CrossRef]
     [Google Scholar]
  16. Koch J. O., Bartenschlager R. 1999; Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B. J Virol 73:7138–7146
     [Google Scholar]
  17. Krieger N., Lohmann V., Bartenschlager R. 2001; Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75:4614–4624 [CrossRef]
     [Google Scholar]
  18. Lan K., Sheu M., Hwang S. 8 other authors 2002; HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis. Oncogene 21:4801–4811 [CrossRef]
     [Google Scholar]
  19. Lin C., Wu J., Hsiao K., Su M. S. 1997; The hepatitis C virus NS4A protein: interaction with the NS4B and NS5A proteins. J Virol 71:6465–6471
     [Google Scholar]
  20. 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]
  21. Lohmann V., Korner F., Dobierzewska A., Bartenschlager R. 2001; Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75:1437–1449 [CrossRef]
     [Google Scholar]
  22. Majumder M., Ghosh A. K., Steele R., Ray R., Ray R. B. 2001; Hepatitis C virus NS5A physically associates with p53 and regulates p21/waf1 gene expression in a p53-dependent manner. J Virol 75:1401–1407 [CrossRef]
     [Google Scholar]
  23. Manns M. P., McHutchison J. G., Gordon S. C. 7 other authors 2001; Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 358:958–965 [CrossRef]
     [Google Scholar]
  24. Murray E. M., Grobler J. A., Markel E. J., Pagnoni M., Paonessa G., Simon A., Flores O. A. 2003; Persistent replication of HCV replicons expressing the beta-lactamase reporter in sub-populations of highly permissive Huh7 cells. J Virol 77:2928–2935 [CrossRef]
     [Google Scholar]
  25. Neddermann P., Clementi A., De Francesco R. 1999; Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein. J Virol 73:9984–9991
     [Google Scholar]
  26. Sambrook J., Fritsch E. F., Maniatis T. 1989 In Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  27. Takeuchi T., Katsume A., Tanaka T., Abe A., Inoue K., Tsukiyama-Kohara K., Kawaguchi R., Tanaka S., Kohara M. 1999; Real-time detection system for quantification of hepatitis C virus genome. Gastroenterology 116:636–642 [CrossRef]
     [Google Scholar]
  28. Tan S., Katze M. G. 2001; How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A. Virology 284:1–12 [CrossRef]
     [Google Scholar]
  29. Tan S., Nakao H., He Y., Vijaysri S., Neddermann P., Jacobs B. L., Mayer B. J., Katze M. G. 1999; NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling. Proc Natl Acad Sci U S A 96:5533–5538 [CrossRef]
     [Google Scholar]
  30. Theodore D., Fried M. W. 2000; Natural history and disease manifestations of hepatitis C infection. Curr Top Microbiol Immunol 242:43–54
     [Google Scholar]
  31. Tu H., Gao L., Shi S. T. 7 other authors 1999; Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein. Virology 263:30–41 [CrossRef]
     [Google Scholar]
  32. World Health Organization 1997; Hepatitis C: global prevalence. Wkly Epidemiol Rec 72:341–344
     [Google Scholar]
  33. Zlokarnik G., Negulescu P. A., Knapp T. E., Mere L., Burres N., Feng L., Whitney M., Roemer K., Tsien R. Y. 1998; Quantitation of transcription and clonal selection of single living cells with β -lactamase as reporter. Science 279:84–88 [CrossRef]
     [Google Scholar]
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Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon
J. Gen. Virol. 85, 1867 (2004); https://doi.org/10.1099/vir.0.80006-0
/content/journal/jgv/10.1099/vir.0.80006-0
/content/journal/jgv/10.1099/vir.0.80006-0
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