1887

Abstract

We have introduced GFP and photoactivatable GFP into the NS5A coding region of a hepatitis C virus (HCV) subgenomic replicon that gives efficient transient replication. NS5A–GFP, expressed by the replicon, could be detected in cytoplasmic fluorescent foci as early as 4 h after RNA was introduced into cells. The fluorescent foci are likely to be sites where RNA synthesis could occur, although their production was not dependent on prior replication. Photobleaching studies demonstrated that the fluorescent proteins were relatively immobile upon expression from replicon RNAs. By contrast, an NS5A–GFP chimera produced in the absence of other viral proteins was mobile. Hence, interactions in cells expressing HCV replication proteins limit NS5A mobility, and transfer of viral proteins between foci is either slow or does not occur. Thus, the sites of HCV RNA replication possibly have a fixed complement of proteins that may act as discrete factories for producing viral RNA.

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2007-02-01
2024-12-01
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References

  1. Appel N., Herian U., Bartenschlager R. 2005a; Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A. J Virol 79:896–909 [CrossRef]
    [Google Scholar]
  2. Appel N., Pietschmann T., Bartenschlager R. 2005b; Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J Virol 79:3187–3194 [CrossRef]
    [Google Scholar]
  3. 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]
  4. Brass V., Bieck E., Montserret R., Wolk B., Hellings J. A., Blum H. E., Penin F., Moradpour D. 2002; An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J Biol Chem 277:8130–8139 [CrossRef]
    [Google Scholar]
  5. 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]
  6. Gao L., Aizaki H., He J. W., Lai M. M. 2004; Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft. J Virol 78:3480–3488 [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. Gretton S. N., Taylor A. I., McLauchlan J. 2005; Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci. J Gen Virol 86:1415–1421 [CrossRef]
    [Google Scholar]
  9. Hamamoto I., Nishimura Y., Okamoto T., Aizaki H., Liu M., Mori Y., Abe T., Suzuki T., Lai M. M. other authors 2005; Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B. J Virol 79:13473–13482 [CrossRef]
    [Google Scholar]
  10. Huang L., Hwang J., Sharma S. D., Hargittai M. R., Chen Y., Arnold J. J., Raney K. D., Cameron C. E. 2005; Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein. J Biol Chem 280:36417–36428 [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. 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]
  13. Lan K. H., Sheu M. L., Hwang S. J., Yen S. H., Chen S. Y., Wu J. C., Wang Y. J., Kato N., Omata M. other authors 2002; HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis. Oncogene 21:4801–4811 [CrossRef]
    [Google Scholar]
  14. Lim S. G., Tan Y. J., Goh P. Y., Lim S. P., Hong W. J. 2006; Use of an in vitro model and yeast two-hybrid system to investigate the pathogenesis of hepatitis C. Intervirology 49:44–50 [CrossRef]
    [Google Scholar]
  15. Liu S., Ansari I. H., Das S. C., Pattnaik A. K. 2006; Insertion and deletion analyses identify regions of non-structural protein 5A of hepatitis C virus that are dispensable for viral genome replication. J Gen Virol 87:323–327 [CrossRef]
    [Google Scholar]
  16. Lohmann V., Korner F., Koch J., 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]
  17. 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]
  18. Macdonald A., Crowder K., Street A., McCormick C., Saksela K., Harris M. 2003; The hepatitis C virus non-structural NS5A protein inhibits activating protein-1 function by perturbing ras-ERK pathway signaling. J Biol Chem 278:17775–17784 [CrossRef]
    [Google Scholar]
  19. Macdonald A., Crowder K., Street A., McCormick C., Harris M. 2004; The hepatitis C virus NS5A protein binds to members of the Src family of tyrosine kinases and regulates kinase activity. J Gen Virol 85:721–729 [CrossRef]
    [Google Scholar]
  20. McCormick C. J., Brown D., Griffin S., Challinor L., Rowlands D. J., Harris M. 2006a; A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A. J Gen Virol 87:93–102 [CrossRef]
    [Google Scholar]
  21. McCormick C. J., Maucourant S., Griffin S., Rowlands D. J., Harris M. 2006b; Tagging of NS5A expressed from a functional hepatitis C virus replicon. J Gen Virol 87:635–640 [CrossRef]
    [Google Scholar]
  22. Miyamoto M., Kato T., Date T., Mizokami M., Wakita T. 2006; Comparison between subgenomic replicons of hepatitis C virus genotypes 2a (JFH-1) and 1b (Con1 NK5.1). Intervirology 49:37–43 [CrossRef]
    [Google Scholar]
  23. Moradpour D., Evans M. J., Gosert R., Yuan Z., Blum H. E., Goff S. P., Lindenbach B. D., Rice C. M. 2004; Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes. J Virol 78:7400–7409 [CrossRef]
    [Google Scholar]
  24. Nanda S. K., Herion D., Liang T. J. 2006; Src homology 3 domain of hepatitis C virus NS5A protein interacts with Bin1 and is important for apoptosis and infectivity. Gastroenterology 130:794–809 [CrossRef]
    [Google Scholar]
  25. Park K. J., Choi S. H., Choi D. H., Park J. M., Yie S. W., Lee S. Y., Hwang S. B. 2003; Hepatitis C virus NS5A protein modulates c-Jun N-terminal kinase through interaction with tumor necrosis factor receptor-associated factor 2. J Biol Chem 278:30711–30718 [CrossRef]
    [Google Scholar]
  26. Patterson G. H., Lippincott-Schwartz J. 2002; A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877 [CrossRef]
    [Google Scholar]
  27. Patterson G. H., Lippincott-Schwartz J. 2004; Selective photolabeling of proteins using photoactivatable GFP. Methods 32:445–450 [CrossRef]
    [Google Scholar]
  28. Shirota Y., Luo H., Qin W., Kaneko S., Yamashita T., Kobayashi K., Murakami S. 2002; Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. J Biol Chem 277:11149–11155 [CrossRef]
    [Google Scholar]
  29. Stow N. D., Hammarsten O., Arbuckle M. I., Elias P. 1993; Inhibition of herpes simplex virus type 1 DNA replication by mutant forms of the origin-binding protein. Virology 196:413–418 [CrossRef]
    [Google Scholar]
  30. Targett-Adams P., McLauchlan J. 2005; Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon. J Gen Virol 86:3075–3080 [CrossRef]
    [Google Scholar]
  31. Wang C., Gale M. Jr, Keller B. C., Huang H., Brown M. S., Goldstein J. L., Ye J. 2005; Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus RNA replication. Mol Cell 18:425–434 [CrossRef]
    [Google Scholar]
  32. Windisch M. P., Frese M., Kaul A., Trippler M., Lohmann V., Bartenschlager R. 2005; Dissecting the interferon-induced inhibition of hepatitis C virus replication by using a novel host cell line. J Virol 79:13778–13793 [CrossRef]
    [Google Scholar]
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