Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1 was unaffected by the presence of neutralizing antibodies that inhibit E2–CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission.


Article metrics loading...

Loading full text...

Full text loading...



  1. Barhoumi, R., Bowen, J. A., Stein, L. S., Echols, J. & Burghardt, R. C.(1993). Concurrent analysis of intracellular glutathione content and gap junctional intercellular communication. Cytometry 14, 747–756.[CrossRef] [Google Scholar]
  2. Bartosch, B., Dubuisson, J. & Cosset, F. L.(2003). Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes. J Exp Med 197, 633–642.[CrossRef] [Google Scholar]
  3. Bazou, D., Dowthwaite, G. P., Khan, I. M., Archer, C. W., Ralphs, J. R. & Coakley, W. T.(2006). Gap junctional intercellular communication and cytoskeletal organization in chondrocytes in suspension in an ultrasound trap. Mol Membr Biol 23, 195–205.[CrossRef] [Google Scholar]
  4. 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]
  5. Carruba, G., Cocciadiferro, L., Bellavia, V., Rizzo, S., Tsatsanis, C., Spandidos, D., Muti, P., Smith, C., Mehta, P. & Castagnetta, L.(2004). Intercellular communication and human hepatocellular carcinoma. Ann N Y Acad Sci 1028, 202–212.[CrossRef] [Google Scholar]
  6. Clayton, R. F., Owsianka, A., Aitken, J., Graham, S., Bhella, D. & Patel, A. H.(2002). Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles. J Virol 76, 7672–7682.[CrossRef] [Google Scholar]
  7. Clayton, R. F., Rinaldi, A., Kandyba, E. E., Edward, M., Willberg, C., Klenerman, P. & Patel, A. H.(2005). Liver cell lines for the study of hepatocyte functions and immunological response. Liver Int 25, 389–402.[CrossRef] [Google Scholar]
  8. Cormier, E. G., Tsamis, F., Kajumo, F., Durso, R. J., Gardner, J. P. & Dragic, T.(2004). CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci U S A 101, 7270–7274.[CrossRef] [Google Scholar]
  9. de Feijter-Rupp, H. L., Hayashi, T., Kalimi, G. H., Edwards, P., Redpath, J. L., Chang, C. C., Stanbridge, E. J. & Trosko, J. E.(1998). Restored gap junctional communication in non-tumorigenic HeLa-normal human fibroblast hybrids. Carcinogenesis 19, 747–754.[CrossRef] [Google Scholar]
  10. Drummer, H. E., Boo, I., Maerz, A. L. & Poumbourios, P.(2006). A conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr motif in hepatitis C virus glycoprotein E2 is a determinant of CD81 binding and viral entry. J Virol 80, 7844–7853.[CrossRef] [Google Scholar]
  11. Friebe, P., Boudet, J., Simorre, J. P. & Bartenschlager, R.(2005). Kissing-loop interaction in the 3′ end of the hepatitis C virus genome essential for RNA replication. J Virol 79, 380–392.[CrossRef] [Google Scholar]
  12. Hadlock, K. G., Lanford, R. E., Perkins, S., Rowe, J., Yang, Q., Levy, S., Pileri, P., Abrignani, S. & Foung, S. K.(2000). Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes. J Virol 74, 10407–10416.[CrossRef] [Google Scholar]
  13. He, T., Tang, C., Liu, Y., Ye, Z., Wu, X., Wei, Y., Moyana, T. & Xiang, J.(2007). Bidirectional membrane molecule transfer between dendritic and T cells. Biochem Biophys Res Commun 359, 202–208.[CrossRef] [Google Scholar]
  14. Helle, F. & Dubuisson, J.(2008). Hepatitis C virus entry into host cells. Cell Mol Life Sci 65, 100–112.[CrossRef] [Google Scholar]
  15. Jolly, C. & Sattentau, Q. J.(2004). Retroviral spread by induction of virological synapses. Traffic 5, 643–650.[CrossRef] [Google Scholar]
  16. 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]
  17. Keck, Z. Y., Li, T. K., Xia, J., Gal-Tanamy, M., Olson, O., Li, S. H., Patel, A. H., Ball, J. K., Lemon, S. M. & Foung, S. K.(2008). Definition of a conserved immunodominant domain on hepatitis C virus E2 glycoprotein by neutralizing human monoclonal antibodies. J Virol 82, 6061–6066.[CrossRef] [Google Scholar]
  18. Koutsoudakis, G., Herrmann, E., Kallis, S., Bartenschlager, R. & Pietschmann, T.(2007). The level of CD81 cell surface expression is a key determinant for productive entry of hepatitis C virus into host cells. J Virol 81, 588–598.[CrossRef] [Google Scholar]
  19. Kuiken, C., Combet, C., Bukh, J., Shin, I. T., Deleage, G., Mizokami, M., Richardson, R., Sablon, E., Yusim, K. & other authors(2006). A comprehensive system for consistent numbering of HCV sequences, proteins and epitopes. Hepatology 44, 1355–1361.[CrossRef] [Google Scholar]
  20. Law, M., Maruyama, T., Lewis, J., Giang, E., Tarr, A. W., Stamataki, Z., Gastaminza, P., Chisari, F. V., Jones, I. M. & other authors(2008). Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med 14, 25–27.[CrossRef] [Google Scholar]
  21. 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]
  22. MacDonald, M. R., Machlin, E. S., Albin, O. R. & Levy, D. E.(2007). The zinc finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses. J Virol 81, 13509–13518.[CrossRef] [Google Scholar]
  23. 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]
  24. Owsianka, A. M., Timms, J. M., Tarr, A. W., Brown, R. J., Hickling, T. P., Szwejk, A., Bienkowska-Szewczyk, K., Thomson, B. J., Patel, A. H. & Ball, J. K.(2006). Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding. J Virol 80, 8695–8704.[CrossRef] [Google Scholar]
  25. Owsianka, A. M., Tarr, A. W., Keck, Z. Y., Li, T. K., Witteveldt, J., Adair, R., Foung, S. K., Ball, J. K. & Patel, A. H.(2008). Broadly neutralizing human monoclonal antibodies to the hepatitis C virus E2 glycoprotein. J Gen Virol 89, 653–659.[CrossRef] [Google Scholar]
  26. Patel, A. H., Wood, J., Penin, F., Dubuisson, J. & McKeating, J. A.(2000). Construction and characterization of chimeric hepatitis C virus E2 glycoproteins: analysis of regions critical for glycoprotein aggregation and CD81 binding. J Gen Virol 81, 2873–2883. [Google Scholar]
  27. Perotti, M., Mancini, N., Diotti, R. A., Tarr, A. W., Ball, J. K., Owsianka, A., Adair, R., Patel, A. H., Clementi, M. & Burioni, R.(2008). Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. J Virol 82, 1047–1052.[CrossRef] [Google Scholar]
  28. Rothwangl, K. B., Manicassamy, B., Uprichard, S. L. & Rong, L.(2008). Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: putative CD81 binding region 1 is not involved in CD81 binding. Virol J 5, 46[CrossRef] [Google Scholar]
  29. Russell, R. S., Meunier, J. C., Takikawa, S., Faulk, K., Engle, R. E., Bukh, J., Purcell, R. H. & Emerson, S. U.(2008). Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus. Proc Natl Acad Sci U S A 105, 4370–4375.[CrossRef] [Google Scholar]
  30. Sherer, N. M., Lehmann, M. J., Jimenez-Soto, L. F., Horensavitz, C., Pypaert, M. & Mothes, W.(2007). Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission. Nat Cell Biol 9, 310–315.[CrossRef] [Google Scholar]
  31. Stong, B. C., Chang, Q., Ahmad, S. & Lin, X.(2006). A novel mechanism for connexin 26 mutation linked deafness: cell death caused by leaky gap junction hemichannels. Laryngoscope 116, 2205–2210.[CrossRef] [Google Scholar]
  32. Tarr, A. W., Owsianka, A. M., Timms, J. M., McClure, C. P., Brown, R. J., Hickling, T. P., Pietschmann, T., Bartenschlager, R., Patel, A. H. & Ball, J. K.(2006). Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33. Hepatology 43, 592–601.[CrossRef] [Google Scholar]
  33. Timpe, J. M., Stamataki, Z., Jennings, A., Hu, K., Farquhar, M. J., Harris, H. J., Schwarz, A., Desombere, I., Roels, G. L. & other authors(2008). Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology 47, 17–24. [Google Scholar]
  34. 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]
  35. Valli, M. B., Serafino, A., Crema, A., Bertolini, L., Manzin, A., Lanzilli, G., Bosman, C., Iacovacci, S., Giunta, S. & other authors(2006). Transmission in vitro of hepatitis C virus from persistently infected human B-cells to hepatoma cells by cell-to-cell contact. J Med Virol 78, 192–201.[CrossRef] [Google Scholar]
  36. Valli, M. B., Crema, A., Lanzilli, G., Serafino, A., Bertolini, L., Ravagnan, G., Ponzetto, A., Menzo, S., Clementi, M. & Carloni, G.(2007). Molecular and cellular determinants of cell-to-cell transmission of HCV in vitro. J Med Virol 79, 1491–1499.[CrossRef] [Google Scholar]
  37. von Hahn, T. & Rice, C. M.(2008). Hepatitis C virus entry. J Biol Chem 283, 3689–3693.[CrossRef] [Google Scholar]
  38. 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]
  39. Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J.(1997). Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 94, 8738–8743.[CrossRef] [Google Scholar]
  40. Zennou, V., Petit, C., Guetard, D., Nerhbass, U., Montagnier, L. & Charneau, P.(2000). HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101, 173–185.[CrossRef] [Google Scholar]

Data & Media loading...


vol. , part 1, pp. 48 - 58

Determination of anti-CD81 neutralizing concentration, stability and effect of nAbs in co-culture experiments [Single PDF file](245 KB)

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error