The three variable regions of hepatitis C virus (HCV) glycoprotein E2 can be removed simultaneously from the E2 ectodomain (residues 384–661) without affecting folding or CD81 binding. In this study, we show that deletion of hypervariable region (HVR) 2 or the intergenotypic variable region (igVR) in the context of the E1E2 polyprotein eliminates formation of heterodimers, reduces CD81 binding and abolishes virus entry. The replication competence of genomic RNA transcribed from the JFH1 infectious HCV clone was not affected by the HVR1, HVR2 or igVR deletions in transfected Huh7.5 cells. However, infectivity of the resultant cell-culture-derived HCV (HCVcc) was abolished by HVR2 or igVR deletions, while deletion of HVR1 led to a 5- to 10-fold reduction in infectivity. Serial passage of cells transfected with genomes lacking HVR1 generated reverted viruses with wild-type levels of infectivity. Sequencing of viral cDNA obtained after full reversion revealed mutations in E1 (I262L) and E2 (N415D) that were present in 35 and 27 % of clones, respectively. Insertion of N415D into HVR1-deleted HCV genomes conferred wild-type levels of infectivity, while I262L increased infectivity by 2.5-fold. These results suggest that HVR2 and the igVR, but not HVR1, are essential for structural integrity and function of the HCV glycoprotein heterodimer.


Article metrics loading...

Loading full text...

Full text loading...



  1. Bankwitz, D., Steinmann, E., Bitzegeio, J., Ciesek, S., Friesland, M., Herrmann, E., Zeisel, M. B., Baumert, T. F., Keck, Z. Y. & other authors(2010). Hepatitis C virus hypervariable region 1 modulates receptor interactions, conceals the CD81 binding site, and protects conserved neutralizing epitopes. J Virol 84, 5751–5763.[CrossRef] [Google Scholar]
  2. Barth, H., Schafer, C., Adah, M. I., Zhang, F., Linhardt, R. J., Toyoda, H., Kinoshita-Toyoda, A., Toida, T., Van Kuppevelt, T. H. & other authors(2003). Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate. J Biol Chem 278, 41003–41012.[CrossRef] [Google Scholar]
  3. Bartosch, B., Vitelli, A., Granier, C., Goujon, C., Dubuisson, J., Pascale, S., Scarselli, E., Cortese, R., Nicosia, A. & other authors(2003). 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]
  4. 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]
  5. Brazzoli, M., Helenius, A., Foung, S. K., Houghton, M., Abrignani, S. & Merola, M.(2005). Folding and dimerization of hepatitis C virus E1 and E2 glycoproteins in stably transfected CHO cells. Virology 332, 438–453.[CrossRef] [Google Scholar]
  6. Cocquerel, L., Meunier, J. C., Pillez, A., Wychowski, C. & Dubuisson, J.(1998). A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J Virol 72, 2183–2191. [Google Scholar]
  7. Cocquerel, L., Kuo, C. C., Dubuisson, J. & Levy, S.(2003). CD81-dependent binding of hepatitis C virus E1E2 heterodimers. J Virol 77, 10677–10683.[CrossRef] [Google Scholar]
  8. Deleersnyder, V., Pillez, A., Wychowski, C., Blight, K., Xu, J., Hahn, Y. S., Rice, C. M. & Dubuisson, J.(1997). Formation of native hepatitis C virus glycoprotein complexes. J Virol 71, 697–704. [Google Scholar]
  9. Dhillon, S., Witteveldt, J., Gatherer, D., Owsianka, A. M., Zeisel, M. B., Zahid, M. N., Rychlowska, M., Foung, S. K., Baumert, T. F. & other authors(2010). Mutations within a conserved region of the hepatitis C virus E2 glycoprotein that influence virus-receptor interactions and sensitivity to neutralizing antibodies. J Virol 84, 5494–5507.[CrossRef] [Google Scholar]
  10. Drummer, H. E. & Poumbourios, P.(2004). Hepatitis C virus glycoprotein E2 contains a membrane-proximal heptad repeat sequence that is essential for E1E2 glycoprotein heterodimerization and viral entry. J Biol Chem 279, 30066–30072.[CrossRef] [Google Scholar]
  11. Drummer, H. E., Wilson, K. A. & Poumbourios, P.(2002). Identification of the hepatitis C virus E2 glycoprotein binding site on the large extracellular loop of CD81. J Virol 76, 11143–11147.[CrossRef] [Google Scholar]
  12. 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]
  13. Drummer, H. E., Wilson, K. A. & Poumbourios, P.(2005). Determinants of CD81 dimerization and interaction with hepatitis C virus glycoprotein E2. Biochem Biophys Res Commun 328, 251–257.[CrossRef] [Google Scholar]
  14. 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]
  15. Drummer, H. E., Boo, I. & Poumbourios, P.(2007). Mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis C virus glycoprotein E1. J Gen Virol 88, 1144–1148.[CrossRef] [Google Scholar]
  16. Dubuisson, J., Hsu, H. H., Cheung, R. C., Greenberg, H. B., Russell, D. G. & Rice, C. M.(1994). Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. J Virol 68, 6147–6160. [Google Scholar]
  17. Evans, M. J., von Hahn, T., Tscherne, D. M., Syder, A. J., Panis, M., Wolk, B., Hatziioannou, T., McKeating, J. A., Bieniasz, P. D. & other authors(2007). Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446, 801–805.[CrossRef] [Google Scholar]
  18. Farci, P., Shimoda, A., Wong, D., Cabezon, T., De Gioannis, D., Strazzera, A., Shimizu, Y., Shapiro, M., Alter, H. J. & other authors(1996). Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Natl Acad Sci U S A 93, 15394–15399.[CrossRef] [Google Scholar]
  19. Grove, J., Nielsen, S., Zhong, J., Bassendine, M. F., Drummer, H. E., Balfe, P. & McKeating, J. A.(2008). Identification of a residue in hepatitis C virus E2 glycoprotein that determines scavenger receptor BI and CD81 receptor dependency and sensitivity to neutralizing antibodies. J Virol 82, 12020–12029.[CrossRef] [Google Scholar]
  20. 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]
  21. Harris, H. J., Davis, C., Mullins, J. G., Hu, K., Goodall, M., Farquhar, M. J., Mee, C. J., McCaffrey, K., Young, S. & other authors(2010). Claudin association with CD81 defines hepatitis C virus entry. J Biol Chem 285, 21092–21102.[CrossRef] [Google Scholar]
  22. He, J., Choe, S., Walker, R., Di Marzio, P., Morgan, D. O. & Landau, N. R.(1995). Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 69, 6705–6711. [Google Scholar]
  23. Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M., Ohkoshi, S. & Shimotohno, K.(1991). Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun 175, 220–228.[CrossRef] [Google Scholar]
  24. Keck, Z. Y., Op De Beeck, A., Hadlock, K. G., Xia, J., Li, T. K., Dubuisson, J. & Foung, S. K.(2004). Hepatitis C virus E2 has three immunogenic domains containing conformational epitopes with distinct properties and biological functions. J Virol 78, 9224–9232.[CrossRef] [Google Scholar]
  25. 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]
  26. Lavillette, D., Pecheur, 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]
  27. Li, H. F., Huang, C. H., Ai, L. S., Chuang, C. K. & Chen, S. S.(2009). Mutagenesis of the fusion peptide-like domain of hepatitis C virus E1 glycoprotein: involvement in cell fusion and virus entry. J Biomed Sci 16, 89.[CrossRef] [Google Scholar]
  28. 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]
  29. McCaffrey, K., Boo, I., Poumbourios, P. & Drummer, H. E.(2007). Expression and characterization of a minimal hepatitis C virus glycoprotein E2 core domain that retains CD81 binding. J Virol 81, 9584–9590.[CrossRef] [Google Scholar]
  30. McCaffrey, K., Vietheer, P. T., Quinn, C., Owczarek, C. M., Hardy, M., Walker, L., Fabri, L., Scotney, P., Poumbourios, P. & other authors(2009). Characterization of a broadly conserved E2 core domain. In Abstracts of the 16th International Meeting on Hepatitis C Virus and Related Viruses, 3–7 October 2009, Nice, France.
  31. Meertens, L., Bertaux, C. & Dragic, T.(2006). Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles. J Virol 80, 11571–11578.[CrossRef] [Google Scholar]
  32. Michalak, J. P., Wychowski, C., Choukhi, A., Meunier, J. C., Ung, S., Rice, C. M. & Dubuisson, J.(1997). Characterization of truncated forms of hepatitis C virus glycoproteins. J Gen Virol 78, 2299–2306. [Google Scholar]
  33. Morikawa, K., Zhao, Z., Date, T., Miyamoto, M., Murayama, A., Akazawa, D., Tanabe, J., Sone, S. & Wakita, T.(2007). The roles of CD81 and glycosaminoglycans in the adsorption and uptake of infectious HCV particles. J Med Virol 79, 714–723.[CrossRef] [Google Scholar]
  34. 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]
  35. 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. & other authors(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]
  36. Patel, J., Patel, A. H. & McLauchlan, J.(2001). The transmembrane domain of the hepatitis C virus E2 glycoprotein is required for correct folding of the E1 glycoprotein and native complex formation. Virology 279, 58–68.[CrossRef] [Google Scholar]
  37. Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner, A. J., Houghton, M., Rosa, D. & other authors(1998). Binding of hepatitis C virus to CD81. Science 282, 938–941.[CrossRef] [Google Scholar]
  38. Ploss, A., Evans, M. J., Gaysinskaya, V. A., Panis, M., You, H., de Jong, Y. P. & Rice, C. M.(2009). Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457, 882–886.[CrossRef] [Google Scholar]
  39. Reed, L. & Muench, H.(1938). A simple method for estimating fifty percent endpoints. Am J Hyg 27, 493–497. [Google Scholar]
  40. Roccasecca, R., Ansuini, H., Vitelli, A., Meola, A., Scarselli, E., Acali, S., Pezzanera, M., Ercole, B. B., McKeating, J. & other authors(2003). Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2. J Virol 77, 1856–1867.[CrossRef] [Google Scholar]
  41. Scarselli, E., Ansuini, H., Cerino, R., Roccasecca, R. M., Acali, S., Filocamo, G., Traboni, C., Nicosia, A., Cortese, R. & other authors(2002). The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J 21, 5017–5025.[CrossRef] [Google Scholar]
  42. 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]
  43. Witteveldt, J., Evans, M. J., Bitzegeio, J., Koutsoudakis, G., Owsianka, A. M., Angus, A. G., Keck, Z. Y., Foung, S. K., Pietschmann, T. & other authors(2009). CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells. J Gen Virol 90, 48–58.[CrossRef] [Google Scholar]
  44. Zeisel, M. B., Koutsoudakis, G., Schnober, E. K., Haberstroh, A., Blum, H. E., Cosset, F. L., Wakita, T., Jaeck, D., Doffoel, M. & other authors(2007). Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81. Hepatology 46, 1722–1731.[CrossRef] [Google Scholar]

Data & Media loading...

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