1887

Abstract

Cell entry and membrane fusion of the hepatitis C virus (HCV) depend on its envelope glycoproteins E1 and E2. HCV pseudotyped particles (HCVpps) are relevant and popular models to study the early steps of the HCV life cycle. However, no structural characterization of HCVpp has been available so far. Using cryo-transmission electron microscopy (cryo-TEM), providing structural information at nanometric resolution, the molecular details of HCVpps and their fusion with liposomes were studied. Cryo-TEM revealed HCVpps as regular 100 nm spherical structures containing the dense retroviral nucleocapsid surrounded by a lipid bilayer. E1–E2 glycoproteins were not readily visible on the membrane surface. Pseudoparticles bearing the E1–E2 glycoproteins of Semliki forest virus looked similar, whereas avian influenza A virus (fowl plague virus) haemagglutinin/neuraminidase-pseudotyped particles exhibited surface spikes. To further characterize HCVpp structurally, a novel method was designed based on magnetic beads covered with anti-HCV antibodies to enrich the samples with particles containing E1–E2. This strategy efficiently sorted HCVpps, which were then directly observed by cryo-TEM in the presence or absence of liposomes at low or neutral pH. After acidification, HCVpps looked the same as at neutral pH and closely contacted the liposomes. These are the first visualizations of early HCV membrane fusion events at the nanometer scale. Furthermore, fluorimetry analysis revealed a relative resistance of HCVpps regarding their fusion capacity when exposed to low pH. This study therefore brings several new molecular details to HCVpp characterization and this efficient strategy of virion immunosorting with magnetic nanobeads is direct, efficient and adaptable to extensive characterization of any virus at a nanometric resolution.

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2010-08-01
2019-11-18
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References

  1. Bartosch, B. & Cosset, F. L. ( 2006; ). Cell entry of hepatitis C virus. Virology 348, 1–12.[CrossRef]
    [Google Scholar]
  2. Bartosch, B. & Cosset, F. L. ( 2009; ). Studying HCV cell entry using HCV pseudoparticles (HCVpp). Methods Mol Biol 510, 279–293.
    [Google Scholar]
  3. Bartosch, B., Dubuisson, J. & Cosset, F. L. ( 2003a; ). Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes. J Exp Med 197, 633–642.[CrossRef]
    [Google Scholar]
  4. Bartosch, B., Vitelli, A., Granier, C., Goujon, C., Dubuisson, J., Pascale, S., Scarselli, E., Cortese, R., Nicosia, A. & Cosset, F. L. ( 2003b; ). Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem 278, 41624–41630.[CrossRef]
    [Google Scholar]
  5. Blanchard, E., Belouzard, S., Goueslain, L., Wakita, T., Dubuisson, J., Wychowski, C. & Rouille, Y. ( 2006; ). Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 80, 6964–6972.[CrossRef]
    [Google Scholar]
  6. Chapel, C., Garcia, C., Bartosch, B., Roingeard, P., Zitzmann, N., Cosset, F. L., Dubuisson, J., Dwek, R. A., Trepo, C. & other authors ( 2007; ). Reduction of the infectivity of hepatitis C virus pseudoparticles by incorporation of misfolded glycoproteins induced by glucosidase inhibitors. J Gen Virol 88, 1133–1143.[CrossRef]
    [Google Scholar]
  7. Corver, J., Ortiz, A., Allison, S. L., Schalich, J., Heinz, F. X. & Wilschut, J. ( 2000; ). Membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system. Virology 269, 37–46.[CrossRef]
    [Google Scholar]
  8. Dreux, M., Pietschmann, T., Granier, C., Voisset, C., Ricard-Blum, S., Mangeot, P. E., Keck, Z., Foung, S., Vu-Dac, N. & other authors ( 2006; ). High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of the scavenger receptor BI. J Biol Chem 281, 18285–18295.[CrossRef]
    [Google Scholar]
  9. Drummer, H. E., Maerz, A. & Poumbourios, P. ( 2003; ). Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins. FEBS Lett 546, 385–390.[CrossRef]
    [Google Scholar]
  10. Dubochet, J., Adrian, M., Chang, J. J., Homo, J. C., Lepault, J., McDowall, A. W. & Schultz, P. ( 1988; ). Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21, 129–228.[CrossRef]
    [Google Scholar]
  11. Fujiyoshi, Y., Kume, N. P., Sakata, K. & Sato, S. B. ( 1994; ). Fine structure of influenza A virus observed by electron cryo-microscopy. EMBO J 13, 318–326.
    [Google Scholar]
  12. Haid, S., Pietschmann, T. & Pecheur, E. I. ( 2009; ). Low pH-dependent hepatitis C virus membrane fusion depends on E2 integrity, target lipid composition, and density of virus particles. J Biol Chem 284, 17657–17667.[CrossRef]
    [Google Scholar]
  13. Harris, A., Cardone, G., Winkler, D. C., Heymann, J. B., Brecher, M., White, J. M. & Steven, A. C. ( 2006; ). Influenza virus pleiomorphy characterized by cryoelectron tomography. Proc Natl Acad Sci U S A 103, 19123–19127.[CrossRef]
    [Google Scholar]
  14. Hatziioannou, T., Valsesia-Wittmann, S., Russell, S. J. & Cosset, F. L. ( 1998; ). Incorporation of fowl plague virus hemagglutinin into murine leukemia virus particles and analysis of the infectivity of the pseudotyped retroviruses. J Virol 72, 5313–5317.
    [Google Scholar]
  15. Helle, F. & Dubuisson, J. ( 2008; ). Hepatitis C virus entry into host cells. Cell Mol Life Sci 65, 100–112.[CrossRef]
    [Google Scholar]
  16. Helle, F., Goffard, A., Morel, V., Duverlie, G., McKeating, J., Keck, Z. Y., Foung, S., Penin, F., Dubuisson, J. & Voisset, C. ( 2007; ). The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein. J Virol 81, 8101–8111.[CrossRef]
    [Google Scholar]
  17. 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]
  18. Kahl, C. A., Marsh, J., Fyffe, J., Sanders, D. A. & Cornetta, K. ( 2004; ). Human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus. J Virol 78, 1421–1430.[CrossRef]
    [Google Scholar]
  19. Keck, Z. Y., Sung, V. M., Perkins, S., Rowe, J., Paul, S., Liang, T. J., Lai, M. M. & Foung, S. K. ( 2004; ). Human monoclonal antibody to hepatitis C virus E1 glycoprotein that blocks virus attachment and viral infectivity. J Virol 78, 7257–7263.[CrossRef]
    [Google Scholar]
  20. Kielian, M. ( 2006; ). Class II virus membrane fusion proteins. Virology 344, 38–47.[CrossRef]
    [Google Scholar]
  21. Kobayashi, M., Bennett, M. C., Bercot, T. & Singh, I. R. ( 2006; ). Functional analysis of hepatitis C virus envelope proteins, using a cell-cell fusion assay. J Virol 80, 1817–1825.[CrossRef]
    [Google Scholar]
  22. 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]
  23. Krey, T., d'Alayer, J., Kikuti, C. M., Saulnier, A., Damier-Piolle, L., Petitpas, I., Johansson, D. X., Tawar, R. G., Baron, B. & other authors ( 2010; ). The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule. PLoS Pathog 6, e1000762 [CrossRef]
    [Google Scholar]
  24. Lavillette, D., Bartosch, B., Nourrisson, D., Verney, G., Cosset, F. L., Penin, F. & Pécheur, E. I. ( 2006; ). Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes. J Biol Chem 281, 3909–3917.[CrossRef]
    [Google Scholar]
  25. Lavillette, D., Pécheur, E. I., Donot, P., Fresquet, J., Molle, J., Corbau, R., Dreux, M., Penin, F. & Cosset, F. L. ( 2007; ). Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus. J Virol 81, 8752–8765.[CrossRef]
    [Google Scholar]
  26. Lindenbach, B. D. & Rice, C. M. ( 2001; ). Flaviviridae: the viruses and their replication. In Fields Virology, pp. 991–1041. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott-Raven.
  27. 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]
  28. Mancini, E. J., Clarke, M., Gowen, B. E., Rutten, T. & Fuller, S. D. ( 2000; ). Cryo-electron microscopy reveals the functional organization of an enveloped virus, Semliki Forest virus. Mol Cell 5, 255–266.[CrossRef]
    [Google Scholar]
  29. Moradpour, D., Penin, F. & Rice, C. M. ( 2007; ). Replication of hepatitis C virus. Nat Rev Microbiol 5, 453–463.[CrossRef]
    [Google Scholar]
  30. Mukhopadhyay, S., Kim, B. S., Chipman, P. R., Rossmann, M. G. & Kuhn, R. J. ( 2003; ). Structure of West Nile virus. Science 302, 248 [CrossRef]
    [Google Scholar]
  31. Mukhopadhyay, S., Kuhn, R. J. & Rossmann, M. G. ( 2005; ). A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3, 13–22.[CrossRef]
    [Google Scholar]
  32. Op De Beeck, A., Cocquerel, L. & Dubuisson, J. ( 2001; ). Biogenesis of hepatitis C virus envelope glycoproteins. J Gen Virol 82, 2589–2595.
    [Google Scholar]
  33. Op De Beeck, A., Voisset, C., Bartosch, B., Ciczora, Y., Cocquerel, L., Keck, Z., Foung, S., Cosset, F. L. & Dubuisson, J. ( 2004; ). Characterization of functional hepatitis C virus envelope glycoproteins. J Virol 78, 2994–3002.[CrossRef]
    [Google Scholar]
  34. Paredes, A. M., Brown, D. T., Rothnagel, R., Chiu, W., Schoepp, R. J., Johnston, R. E. & Prasad, B. V. ( 1993; ). Three-dimensional structure of a membrane-containing virus. Proc Natl Acad Sci U S A 90, 9095–9099.[CrossRef]
    [Google Scholar]
  35. Sandrin, V. & Cosset, F. L. ( 2006; ). Intracellular versus cell surface assembly of retroviral pseudotypes is determined by the cellular localization of the viral glycoprotein, its capacity to interact with Gag, and the expression of the Nef protein. J Biol Chem 281, 528–542.[CrossRef]
    [Google Scholar]
  36. Smit, J. M., Bittman, R. & Wilschut, J. ( 1999; ). Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes. J Virol 73, 8476–8484.
    [Google Scholar]
  37. Stegmann, T., Hoekstra, D., Scherphof, G. & Wilschut, J. ( 1985; ). Kinetics of pH-dependent fusion between influenza virus and liposomes. Biochemistry 24, 3107–3113.[CrossRef]
    [Google Scholar]
  38. Stegmann, T., Hoekstra, D., Scherphof, G. & Wilschut, J. ( 1986; ). Fusion activity of influenza virus. A comparison between biological and artificial target membrane vesicles. J Biol Chem 261, 10966–10969.
    [Google Scholar]
  39. Stegmann, T., Nir, S. & Wilschut, J. ( 1989; ). Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes. Biochemistry 28, 1698–1704.[CrossRef]
    [Google Scholar]
  40. Strauss, J. H. & Strauss, E. G. ( 2001; ). Virus evolution: how does an enveloped virus make a regular structure? Cell 105, 5–8.[CrossRef]
    [Google Scholar]
  41. Tscherne, D. M., Jones, C. T., Evans, M. J., Lindenbach, B. D., McKeating, J. A. & Rice, C. M. ( 2006; ). Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol 80, 1734–1741.[CrossRef]
    [Google Scholar]
  42. Waarts, B. L., Smit, J. M., Aneke, O. J., McInerney, G. M., Liljestrom, P., Bittman, R. & Wilschut, J. ( 2005; ). Reversible acid-induced inactivation of the membrane fusion protein of Semliki Forest virus. J Virol 79, 7942–7948.[CrossRef]
    [Google Scholar]
  43. 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]
  44. Yu, X., Qiao, M., Atanasov, I., Hu, Z., Kato, T., Liang, T. & Zhou, Z. ( 2007; ). Cryo-electron microscopy and three-dimensional reconstructions of hepatitis C virus particles. Virology 367, 126–134.[CrossRef]
    [Google Scholar]
  45. Zhang, W., Chipman, P. R., Corver, J., Johnson, P. R., Zhang, Y., Mukhopadhyay, S., Baker, T. S., Strauss, J. H., Rossmann, M. G. & Kuhn, R. J. ( 2003; ). Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol 10, 907–912.[CrossRef]
    [Google Scholar]
  46. 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]
  47. Zhu, P., Liu, J., Bess, J., Jr, Chertova, E., Lifson, J. D., Grise, H., Ofek, G. A., Taylor, K. A. & Roux, K. H. ( 2006; ). Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441, 847–852.[CrossRef]
    [Google Scholar]
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