1887

Abstract

p7 protein is a small protein encoded by (HCV) that functions as an ion channel in planar lipid bilayers. The function of p7 is vital for the virus life cycle. In this study, the p7 protein of genotype 2a (strain JFH1; the only strain that replicates and produces virus progeny ) was tagged with either an enhanced green fluorescent protein (eGFP) or a haemagglutinin (HA) epitope to facilitate tracking of the protein in the intracellular environment. The tagged viral polyprotein was expressed transiently in the cells after transfection with the recombinant RNA transcripts. Confocal microscopy revealed that the tagged p7 protein was localized in the endoplasmic reticulum (ER) but not associated with mitochondria. Immunoelectron microscopy confirmed the p7 localization data and, moreover, showed that intracellular virus-like particles formed in the cells transfected with the wild-type, but not the recombinant, transcripts. Following a few passages of the transfected cells, the recombinant genome with the HA tag reverted to wild-type and the entire tag was deleted. Therefore, in this study, it has been demonstrated that the p7 protein in the context of the full-length polyprotein encoded by a replication competent genome is only localized to the ER and has a possible role in HCV particle formation.

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2007-01-01
2024-04-19
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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. Carrere-Kremer S., Montpellier-Pala C., Cocquerel L., Wychowski C., Penin F., Dubuisson J. 2002; Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol 76:3720–3730 [CrossRef]
    [Google Scholar]
  3. Carrere-Kremer S., Montpellier C., Lorenzo L., Brulin B., Cocquerel L., Belouzard S., Penin F., Dubuisson J. 2004; Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions. J Biol Chem 279:41384–41392 [CrossRef]
    [Google Scholar]
  4. Condreay J. P., Witherspoon S. M., Clay W. C., Kost T. A. 1999; Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proc Natl Acad Sci U S A 96:127–132 [CrossRef]
    [Google Scholar]
  5. Fargion S., Fracanzani A. L., Valenti L. 2004; Treatment choices for people infected with HCV. J Antimicrob Chemother 53:708–712 [CrossRef]
    [Google Scholar]
  6. Fischer W. B., Sansom M. S. 2002; Viral ion channels: structure and function. Biochim Biophys Acta 1561:27–45 [CrossRef]
    [Google Scholar]
  7. Gonzalez M. E., Carrasco L. 2003; Viroporins. FEBS Lett 552:28–34 [CrossRef]
    [Google Scholar]
  8. Griffin S. D., Beales L. P., Clarke D. S., Worsfold O., Evans S. D., Jaeger J., Harris M. P., Rowlands D. J. 2003; The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett 535:34–38 [CrossRef]
    [Google Scholar]
  9. Griffin S. D. C., Harvey R., Clarke D. S., Barclay W. S., Harris M., Rowlands D. J. 2004; A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria. J Gen Virol 85:451–461 [CrossRef]
    [Google Scholar]
  10. Griffin S., Clarke D., McCormick C., Rowlands D., Harris M. 2005; Signal peptide cleavage and internal targeting signals direct the hepatitis C virus p7 protein to distinct intracellular membranes. J Virol 79:15525–15536 [CrossRef]
    [Google Scholar]
  11. Houghton M. 1996; Hepatitis C viruses. In Fields Virology . , 3rd edn. pp  1035–1058 Edited by Fields B. N., Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott-Raven;
  12. Huang Y. W., Haqshenas G., Kasorndorkbua C., Halbur P. G., Emerson S. U., Meng X. J. 2005; Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs. J Virol 79:1552–1558 [CrossRef]
    [Google Scholar]
  13. 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]
  14. Koutsoudakis G., Kaul A., Steinmann E., Kallis S., Lohmann V., Pietschmann T., Bartenschlager R. 2006; Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J Virol 80:5308–5320 [CrossRef]
    [Google Scholar]
  15. Lin C., Lindenbach B. D., Pragai B. M., McCourt D. W., Rice C. M. 1994; Processing in the hepatitis C virus E2–NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol 68:5063–5073
    [Google Scholar]
  16. Lindenbach B. D., Evans M. J., Syder A. J., Wolk B., Tellinghuisen T. L., Liu C. C., Maruyama T., Hynes R. O., Burton D. R. other authors 2005; Complete replication of hepatitis C virus in cell culture. Science 309:623–626 [CrossRef]
    [Google Scholar]
  17. Lindenbach B. D., Meuleman P., Ploss A., Vanwolleghem T., Syder A. J., McKeating J. A., Lanford R. E., Feinstone S. M., Major M. E. other authors 2006; Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci U S A 103:3805–3809 [CrossRef]
    [Google Scholar]
  18. 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]
  19. Mackenzie J. M., Westaway E. G. 2001; Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J Virol 75:10787–10799 [CrossRef]
    [Google Scholar]
  20. Nielsen H., Engelbrecht J., Brunak S., von Heijne G. 1997; Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6 [CrossRef]
    [Google Scholar]
  21. Owsianka A., Tarr A. W., Juttla V. S., Lavillette D., Bartosch B., Cosset F. L., Ball J. K., Patel A. H. 2005; Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J Virol 79:11095–11104 [CrossRef]
    [Google Scholar]
  22. Pavlovic D., Neville D. C., Argaud O., Blumberg B., Dwek R. A., Fischer W. B., Zitzmann N. 2003; The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci U S A 100:6104–6108 [CrossRef]
    [Google Scholar]
  23. Pinto L. H., Holsinger L. J., Lamb R. A. 1992; Influenza virus M2 protein has ion channel activity. Cell 69:517–528 [CrossRef]
    [Google Scholar]
  24. Premkumar A., Wilson L., Ewart G. D., Gage P. W. 2004; Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride. FEBS Lett 557:99–103 [CrossRef]
    [Google Scholar]
  25. Rouille Y., Helle F., Delgrange D., Roingeard P., Voisset C., Blanchard E., Belouzard S., McKeating J., Patel A. H. other authors 2006; Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus. J Virol 80:2832–2841 [CrossRef]
    [Google Scholar]
  26. Sakai A., Claire M. S., Faulk K., Govindarajan S., Emerson S. U., Purcell R. H., Bukh J. 2003; The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences. Proc Natl Acad Sci U S A 100:11646–11651 [CrossRef]
    [Google Scholar]
  27. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  28. Takeda M., Pekosz A., Shuck K., Pinto L. H., Lamb R. A. 2002; Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture. J Virol 76:1391–1399 [CrossRef]
    [Google Scholar]
  29. Wakita T., Pietschmann T., Kato T., Date T., Miyamoto M., Zhao Z., Murthy K., Habermann A., Krausslich H. G. 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]
  30. Yi M., Villanueva R. A., Thomas D. L., Wakita T., Lemon S. M. 2006; Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci U S A 103:2310–2315 [CrossRef]
    [Google Scholar]
  31. Zhong J., Gastaminza P., Cheng G., Kapadia S., Kato T., Burton D. R., Wieland S. F., Uprichard S. L., Wakita T., Chisari F. V. 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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