1887

Abstract

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted -membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

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2004-02-01
2019-11-18
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References

  1. Aldabe, R. & Carrasco, L. ( 1995; ). Induction of membrane proliferation by poliovirus proteins 2C and 2BC. Biochem Biophys Res Commun 206, 64–76.[CrossRef]
    [Google Scholar]
  2. Berg, T., Kronenberger, B., Hinrichsen, H. & 10 other authors ( 2003; ). Triple therapy with amantadine in treatment-naive patients with chronic hepatitis C: a placebo-controlled trial. Hepatology 37, 1359–1367.[CrossRef]
    [Google Scholar]
  3. Boya, P., Roques, B. & Kroemer, G. ( 2001; ). New EMBO members' review: viral and bacterial proteins regulating apoptosis at the mitochondrial level. EMBO J 20, 4325–4331.[CrossRef]
    [Google Scholar]
  4. Boya, P., Roumier, T., Andreau, K., Gonzalez-Polo, R. A., Zamzami, N., Castedo, M. & Kroemer, G. ( 2003; ). Mitochondrion-targeted apoptosis regulators of viral origin. Biochem Biophys Res Commun 304, 575–581.[CrossRef]
    [Google Scholar]
  5. Brillanti, S., Levantesi, F., Masi, L., Foli, M. & Bolondi, L. ( 2000; ). Triple antiviral therapy as a new option for patients with interferon nonresponsive chronic hepatitis C. Hepatology 32, 630–634.[CrossRef]
    [Google Scholar]
  6. 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]
  7. Choo, Q. L., Kuo, G., Weiner, A., Wang, K. S., Overby, L., Bradley, D. & Houghton, M. ( 1992; ). Identification of the major, parenteral non-A, non-B hepatitis agent (hepatitis C virus) using a recombinant cDNA approach. Semin Liver Dis 12, 279–288.[CrossRef]
    [Google Scholar]
  8. Ciampor, F., Bayley, P. M., Nermut, M. V., Hirst, E. M., Sugrue, R. J. & Hay, A. J. ( 1992; ). Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans Golgi compartment. Virology 188, 14–24.[CrossRef]
    [Google Scholar]
  9. Clarke, B. ( 1997; ). Molecular virology of hepatitis C virus. J Gen Virol 78, 2397–2410.
    [Google Scholar]
  10. Cozzolongo, R., Cuppone, R. & Manghisi, O. G. ( 2002; ). The treatment of chronic hepatitis C not responding to interferon. Curr Pharm Des 8, 967–975.[CrossRef]
    [Google Scholar]
  11. D'Agostino, D. M., Ranzato, L., Arrigoni, G. & 10 other authors ( 2002; ). Mitochondrial alterations induced by the p13II protein of human T-cell leukemia virus type 1. Critical role of arginine residues. J Biol Chem 277, 34424–34433.[CrossRef]
    [Google Scholar]
  12. Duff, K. C. & Ashley, R. H. ( 1992; ). The transmembrane domain of influenza A M2 protein forms amantadine-sensitive proton channels in planar lipid bilayers. Virology 190, 485–489.[CrossRef]
    [Google Scholar]
  13. Duff, K. C., Gilchrist, P. J., Saxena, A. M. & Bradshaw, J. P. ( 1994; ). Neutron diffraction reveals the site of amantadine blockade in the influenza A M2 ion channel. Virology 202, 287–293.[CrossRef]
    [Google Scholar]
  14. 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]
  15. Ewart, G. D., Sutherland, T., Gage, P. W. & Cox, G. B. ( 1996; ). The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels. J Virol 70, 7108–7115.
    [Google Scholar]
  16. Gibbs, J. S., Malide, D., Hornung, F., Bennink, J. R. & Yewdell, J. W. ( 2003; ). The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function. J Virol 77, 7214–7224.[CrossRef]
    [Google Scholar]
  17. Gonzalez, M. E. & Carrasco, L. ( 1998; ). The human immunodeficiency virus type 1 Vpu protein enhances membrane permeability. Biochemistry 37, 13710–13719.[CrossRef]
    [Google Scholar]
  18. 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]
  19. Halestrap, A. P. & Brennerb, C. ( 2003; ). The adenine nucleotide translocase: a central component of the mitochondrial permeability transition pore and key player in cell death. Curr Med Chem 10, 1507–1525.[CrossRef]
    [Google Scholar]
  20. Harada, T., Tautz, N. & Thiel, H. J. ( 2000; ). E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J Virol 74, 9498–9506.[CrossRef]
    [Google Scholar]
  21. Hay, A. J., Wolstenholme, A. J., Skehel, J. J. & Smith, M. H. ( 1985; ). The molecular basis of the specific anti-influenza action of amantadine. EMBO J 4, 3021–3024.
    [Google Scholar]
  22. Hsu, M., Zhang, J., Flint, M., Logvinoff, C., Cheng-Mayer, C., Rice, C. M. & McKeating, J. A. ( 2003; ). Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci U S A 100, 7271–7276.[CrossRef]
    [Google Scholar]
  23. Jacotot, E., Ferri, K. F., El Hamel, C. & 17 other authors ( 2001; ). Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2. J Exp Med 193, 509–519.[CrossRef]
    [Google Scholar]
  24. Jubin, R., Murray, M. G., Howe, A. Y., Butkiewicz, N., Hong, Z. & Lau, J. Y. ( 2000; ). Amantadine and rimantadine have no direct inhibitory effects against hepatitis C viral protease, helicase, ATPase, polymerase, and internal ribosomal entry site-mediated translation. J Infect Dis 181, 331–334.[CrossRef]
    [Google Scholar]
  25. Kunkel, T. A., Bebenek, K. & McClary, J. ( 1991; ). Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol 204, 125–139.
    [Google Scholar]
  26. Lamb, R. A. & Pinto, L. H. ( 1997; ). Do Vpu and Vpr of human immunodeficiency virus type 1 and NB of influenza B virus have ion channel activities in the viral life cycles? Virology 229, 1–11.[CrossRef]
    [Google Scholar]
  27. Liljestrom, P., Lusa, S., Huylebroeck, D. & Garoff, H. ( 1991; ). In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol 65, 4107–4113.
    [Google Scholar]
  28. 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]
  29. Loewy, A., Smyth, J., von Bonsdorff, C. H., Liljestrom, P. & Schlesinger, M. J. ( 1995; ). The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process. J Virol 69, 469–475.
    [Google Scholar]
  30. Martin, J., Navas, S., Fernandez, M., Rico, M., Pardo, M., Quiroga, J. A., Zahm, F. & Carreno, V. ( 1999; ). In vitro effect of amantadine and interferon alpha-2a on hepatitis C virus markers in cultured peripheral blood mononuclear cells from hepatitis C virus-infected patients. Antivir Res 42, 59–70.[CrossRef]
    [Google Scholar]
  31. Medeiros, R., Escriou, N., Naffakh, N., Manuguerra, J. C. & van der Werf, S. ( 2001; ). Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes. Virology 289, 74–85.[CrossRef]
    [Google Scholar]
  32. Meyers, G., Tautz, N., Becher, P., Thiel, H. J. & Kummerer, B. M. ( 1996; ). Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs. J Virol 70, 8606–8613.
    [Google Scholar]
  33. Mizushima, H., Hijikata, M., Asabe, S., Hirota, M., Kimura, K. & Shimotohno, K. ( 1994; ). Two hepatitis C virus glycoprotein E2 products with different C termini. J Virol 68, 6215–6222.
    [Google Scholar]
  34. Mottola, G., Cardinali, G., Ceccacci, A., Trozzi, C., Bartholomew, L., Torrisi, M. R., Pedrazzini, E., Bonatti, S. & Migliaccio, G. ( 2002; ). Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293, 31–43.[CrossRef]
    [Google Scholar]
  35. Newton, K., Meyer, J. C., Bellamy, A. R. & Taylor, J. A. ( 1997; ). Rotavirus nonstructural glycoprotein NSP4 alters plasma membrane permeability in mammalian cells. J Virol 71, 9458–9465.
    [Google Scholar]
  36. Ohuchi, M., Cramer, A., Vey, M., Ohuchi, R., Garten, W. & Klenk, H. D. ( 1994; ). Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein. J Virol 68, 920–926.
    [Google Scholar]
  37. Okada, A., Miura, T. & Takeuchi, H. ( 2001; ). Protonation of histidine and histidine-tryptophan interaction in the activation of the M2 ion channel from influenza a virus. Biochemistry 40, 6053–6060.[CrossRef]
    [Google Scholar]
  38. 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]
  39. Piai, G., Rocco, P., Tartaglione, M. T. & 7 other authors ( 2003; ). Triple (interferon, ribavirin, amantadine) versus double (interferon, ribavirin) re-therapy for interferon relapser genotype 1b HCV chronic active hepatitis patients. Hepatol Res 25, 355–363.[CrossRef]
    [Google Scholar]
  40. Piller, S. C., Ewart, G. D., Premkumar, A., Cox, G. B. & Gage, P. W. ( 1996; ). Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers. Proc Natl Acad Sci U S A 93, 111–115.[CrossRef]
    [Google Scholar]
  41. Piller, S. C., Ewart, G. D., Jans, D. A., Gage, P. W. & Cox, G. B. ( 1999; ). The amino-terminal region of Vpr from human immunodeficiency virus type 1 forms ion channels and kills neurons. J Virol 73, 4230–4238.
    [Google Scholar]
  42. 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]
  43. Rahmani, Z., Huh, K. W., Lasher, R. & Siddiqui, A. ( 2000; ). Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential. J Virol 74, 2840–2846.[CrossRef]
    [Google Scholar]
  44. Robertson, B., Myers, G., Howard, C. & 14 other authors ( 1998; ). Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. International Committee on Virus Taxonomy. Arch Virol 143, 2493–2503.[CrossRef]
    [Google Scholar]
  45. Sakaguchi, T., Leser, G. P. & Lamb, R. A. ( 1996; ). The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus. J Cell Biol 133, 733–747.[CrossRef]
    [Google Scholar]
  46. 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]
  47. Salom, D., Hill, B. R., Lear, J. D. & DeGrado, W. F. ( 2000; ). pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus. Biochemistry 39, 14160–14170.[CrossRef]
    [Google Scholar]
  48. Saunier, B., Triyatni, M., Ulianich, L., Maruvada, P., Yen, P. & Kohn, L. D. ( 2003; ). Role of the asialoglycoprotein receptor in binding and entry of hepatitis C virus structural proteins in cultured human hepatocytes. J Virol 77, 546–559.[CrossRef]
    [Google Scholar]
  49. Schmidt-Mende, J., Bieck, E., Hugle, T., Penin, F., Rice, C. M., Blum, H. E. & Moradpour, D. ( 2001; ). Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 276, 44052–44063.[CrossRef]
    [Google Scholar]
  50. Takada, S., Shirakata, Y., Kaneniwa, N. & Koike, K. ( 1999; ). Association of hepatitis B virus X protein with mitochondria causes mitochondrial aggregation at the nuclear periphery, leading to cell death. Oncogene 18, 6965–6973.[CrossRef]
    [Google Scholar]
  51. Takeuchi, K. & Lamb, R. A. ( 1994; ). Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport. J Virol 68, 911–919.
    [Google Scholar]
  52. Thuluvath, P. J., Pande, H. & Maygers, J. ( 2003; ). Combination therapy with interferon-alpha(2b), ribavirin, and amantadine in chronic hepatitis C nonresponders to interferon and ribavirin. Dig Dis Sci 48, 594–597.[CrossRef]
    [Google Scholar]
  53. Tian, P., Estes, M. K., Hu, Y., Ball, J. M., Zeng, C. Q. & Schilling, W. P. ( 1995; ). The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum. J Virol 69, 5763–5772.
    [Google Scholar]
  54. van Kuppeveld, F. J., Hoenderop, J. G., Smeets, R. L., Willems, P. H., Dijkman, H. B., Galama, J. M. & Melchers, W. J. ( 1997; ). Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release. EMBO J 16, 3519–3532.[CrossRef]
    [Google Scholar]
  55. Wang, C., Lamb, R. A. & Pinto, L. H. ( 1995; ). Activation of the M2 ion channel of influenza virus: a role for the transmembrane domain histidine residue. Biophys J 69, 1363–1371.[CrossRef]
    [Google Scholar]
  56. Weegink, C. J., Sentjens, R. E., Beld, M. G., Dijkgraaf, M. G. & Reesink, H. W. ( 2003; ). Chronic hepatitis C patients with a post-treatment virological relapse re-treated with an induction dose of 18 MU interferon-alpha in combination with ribavirin and amantadine: a two-arm randomized pilot study. J Viral Hepat 10, 174–182.[CrossRef]
    [Google Scholar]
  57. Yanagi, M., St Claire, M., Shapiro, M., Emerson, S. U., Purcell, R. H. & Bukh, J. ( 1998; ). Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 244, 161–172.[CrossRef]
    [Google Scholar]
  58. Zilly, M., Lingenauber, C., Desch, S., Vath, T., Klinker, H. & Langmann, P. ( 2002; ). Triple antiviral re-therapy for chronic hepatitis C with interferon-alpha, ribavirin and amantadine in nonresponders to interferon-alpha and ribavirin. Eur J Med Res 7, 149–154.
    [Google Scholar]
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