1887

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Volume 92, Issue 10

Comprehensive linker-scanning mutagenesis of the hepatitis C virus E1 and E2 envelope glycoproteins reveals new structure–function relationships Open Access

Abstract

Despite extensive research, many details about the structure and functions of hepatitis C virus (HCV) glycoproteins E1 and E2 are not fully understood, and their crystal structure remains to be determined. We applied linker-scanning mutagenesis to generate a panel of 34 mutants, each containing an insertion of 5 aa at a random position within the E1E2 sequence. The mutated glycoproteins were analysed by using a range of assays to identify regions critical for maintaining protein conformation, E1E2 complex assembly, CD81 receptor binding, membrane fusion and infectivity. The results, while supporting previously published data, provide several interesting new findings. Firstly, insertion at amino acid 587 or 596 reduced E1E2 heterodimerization without affecting reactivity with some conformation-sensitive mAbs or with CD81, thus implicating these residues in glycoprotein assembly. Secondly, insertions within a conserved region of E2, between amino acid residues 611 and 631, severely disrupted protein conformation and abrogated binding of all conformation-sensitive antibodies, suggesting that the structural integrity of this region is critical for the correct folding of E2. Thirdly, an insertion at Leu-682 specifically affected membrane fusion, providing direct evidence that the membrane-proximal ‘stem’ of E2 is involved in the fusion mechanism. Overall, our results show that the HCV glycoproteins generally do not tolerate insertions and that there are a very limited number of sites that can be changed without dramatic loss of function. Nevertheless, we identified two E2 insertion mutants, at amino acid residues 408 and 577, that were infectious in the murine leukemia virus-based HCV pseudoparticle system.

  • Received:
  • Accepted:
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© 2011 SGM
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2011-10-01
2024-04-19
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References

  1. Arumugaswami V., Remenyi R., Kanagavel V., Sue E. Y., Ngoc Ho T., Liu C., Fontanes V., Dasgupta A., Sun R. 2008; High-resolution functional profiling of hepatitis C virus genome. PLoS Pathog 4:e1000182 [View Article][PubMed]
     [Google Scholar]
  2. Bankwitz D., Steinmann E., Bitzegeio J., Ciesek S., Friesland M., Herrmann E., Zeisel M. B., Baumert T. F., Keck Z. Y. et al. 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 [View Article][PubMed]
     [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 [View Article][PubMed]
     [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 [View Article][PubMed]
     [Google Scholar]
  5. Benedicto I., Molina-Jiménez F., Barreiro O., Maldonado-Rodríguez A., Prieto J., Moreno-Otero R., Aldabe R., López-Cabrera M., Majano P. L. 2008; Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum. Hepatology 48:1044–1053 [View Article][PubMed]
     [Google Scholar]
  6. Blanchard E., Belouzard S., Goueslain L., Wakita T., Dubuisson J., Wychowski C., Rouillé Y. 2006; Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 80:6964–6972 [View Article][PubMed]
     [Google Scholar]
  7. Brass V., Moradpour D., Blum H. E. 2006; Molecular virology of hepatitis C virus (HCV): 2006 update. Int J Med Sci 3:29–34[PubMed] [CrossRef]
     [Google Scholar]
  8. Burlone M. E., Budkowska A. 2009; Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J Gen Virol 90:1055–1070 [View Article][PubMed]
     [Google Scholar]
  9. Ciczora Y., Callens N., Montpellier C., Bartosch B., Cosset F. L., Op de Beeck A., Dubuisson J. 2005; Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer. J Gen Virol 86:2793–2798 [View Article][PubMed]
     [Google Scholar]
  10. 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 [View Article][PubMed]
     [Google Scholar]
  11. 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[PubMed]
     [Google Scholar]
  12. Cocquerel L., Wychowski C., Minner F., Penin F., Dubuisson J. 2000; Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins. J Virol 74:3623–3633 [View Article][PubMed]
     [Google Scholar]
  13. Cocquerel L., Kuo C. C., Dubuisson J., Levy S. 2003; CD81-dependent binding of hepatitis C virus E1E2 heterodimers. J Virol 77:10677–10683 [View Article][PubMed]
     [Google Scholar]
  14. 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 [View Article][PubMed]
     [Google Scholar]
  15. 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 [View Article][PubMed]
     [Google Scholar]
  16. 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 [View Article][PubMed]
     [Google Scholar]
  17. Dubuisson J. 2007; Hepatitis C virus proteins. World J Gastroenterol 13:2406–2415[PubMed] [CrossRef]
     [Google Scholar]
  18. 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[PubMed]
     [Google Scholar]
  19. Forns X., Thimme R., Govindarajan S., Emerson S. U., Purcell R. H., Chisari F. V., Bukh J. 2000; Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees. Proc Natl Acad Sci U S A 97:13318–13323 [View Article][PubMed]
     [Google Scholar]
  20. Goffard A., Callens N., Bartosch B., Wychowski C., Cosset F. L., Montpellier C., Dubuisson J. 2005; Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins. J Virol 79:8400–8409 [View Article][PubMed]
     [Google Scholar]
  21. 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 [View Article][PubMed]
     [Google Scholar]
  22. Harrison S. C. 2008; Viral membrane fusion. Nat Struct Mol Biol 15:690–698 [View Article][PubMed]
     [Google Scholar]
  23. Helle F., Dubuisson J. 2008; Hepatitis C virus entry into host cells. Cell Mol Life Sci 65:100–112 [View Article][PubMed]
     [Google Scholar]
  24. 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 [View Article][PubMed]
     [Google Scholar]
  25. Humphreys I., Fleming V., Fabris P., Parker J., Schulenberg B., Brown A., Demetriou C., Gaudieri S., Pfafferott K. et al. other authors 2009; Full-length characterization of hepatitis C virus subtype 3a reveals novel hypervariable regions under positive selection during acute infection. J Virol 83:11456–11466 [View Article][PubMed]
     [Google Scholar]
  26. Iacob S., Gheorghe L., Hrehoret D., Becheanu G., Herlea V., Popescu I. 2008; Pegylated interferon α-2a and ribavirin combination therapy in HCV liver transplant recipients. Experience of 7 cases. J Gastrointestin Liver Dis 17:165–172[PubMed]
     [Google Scholar]
  27. Ikeda Y., Takeuchi Y., Martin F., Cosset F. L., Mitrophanous K., Collins M. 2003; Continuous high-titer HIV-1 vector production. Nat Biotechnol 21:569–572 [View Article][PubMed]
     [Google Scholar]
  28. Keck Z. Y., Op De Beeck A., Hadlock K. G., Xia J., Li T. K., Dubuisson J., Foung S. K. 2004a; Hepatitis C virus E2 has three immunogenic domains containing conformational epitopes with distinct properties and biological functions. J Virol 78:9224–9232 [View Article][PubMed]
     [Google Scholar]
  29. Keck Z. Y., Sung V. M., Perkins S., Rowe J., Paul S., Liang T. J., Lai M. M., Foung S. K. 2004b; Human monoclonal antibody to hepatitis C virus E1 glycoprotein that blocks virus attachment and viral infectivity. J Virol 78:7257–7263 [View Article][PubMed]
     [Google Scholar]
  30. Keck Z. Y., Machida K., Lai M. M., Ball J. K., Patel A. H., Foung S. K. 2008; Therapeutic control of hepatitis C virus: the role of neutralizing monoclonal antibodies. Curr Top Microbiol Immunol 317:1–38 [View Article][PubMed]
     [Google Scholar]
  31. Kielian M., Rey F. A. 2006; Virus membrane-fusion proteins: more than one way to make a hairpin. Nat Rev Microbiol 4:67–76 [View Article][PubMed]
     [Google Scholar]
  32. 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 [View Article][PubMed]
     [Google Scholar]
  33. Krey T., d’Alayer J., Kikuti C. M., Saulnier A., Damier-Piolle L., Petitpas I., Johansson D. X., Tawar R. G., Baron B. et al. other authors 2010; The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule. PLoS Pathog 6:e1000762 [View Article][PubMed]
     [Google Scholar]
  34. Lavie M., Goffard A., Dubuisson J. 2007; Assembly of a functional HCV glycoprotein heterodimer. Curr Issues Mol Biol 9:71–86[PubMed]
     [Google Scholar]
  35. 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 [View Article][PubMed]
     [Google Scholar]
  36. 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 [View Article][PubMed]
     [Google Scholar]
  37. 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 [View Article][PubMed]
     [Google Scholar]
  38. 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[PubMed]
     [Google Scholar]
  39. Op De Beeck A., Montserret R., Duvet S., Cocquerel L., Cacan R., Barberot B., Le Maire M., Penin F., Dubuisson J. 2000; The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization. J Biol Chem 275:31428–31437 [View Article][PubMed]
     [Google Scholar]
  40. Owsianka A., Clayton R. F., Loomis-Price L. D., McKeating J. A., Patel A. H. 2001; Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2. J Gen Virol 82:1877–1883[PubMed]
     [Google Scholar]
  41. 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 [View Article][PubMed]
     [Google Scholar]
  42. 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 [View Article][PubMed]
     [Google Scholar]
  43. 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 [View Article][PubMed]
     [Google Scholar]
  44. 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[PubMed]
     [Google Scholar]
  45. 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 [View Article][PubMed]
     [Google Scholar]
  46. Pérez-Berná A. J., Moreno M. R., Guillén J., Bernabeu A., Villalaín J. 2006; The membrane-active regions of the hepatitis C virus E1 and E2 envelope glycoproteins. Biochemistry 45:3755–3768 [View Article][PubMed]
     [Google Scholar]
  47. Pileri P., Uematsu Y., Campagnoli S., Galli G., Falugi F., Petracca R., Weiner A. J., Houghton M., Rosa D. et al. other authors 1998; Binding of hepatitis C virus to CD81. Science 282:938–941 [View Article][PubMed]
     [Google Scholar]
  48. 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 [View Article][PubMed]
     [Google Scholar]
  49. Roccasecca R., Ansuini H., Vitelli A., Meola A., Scarselli E., Acali S., Pezzanera M., Ercole B. B., McKeating J. et al. 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 [View Article][PubMed]
     [Google Scholar]
  50. 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 [View Article][PubMed]
     [Google Scholar]
  51. Russell R. S., Kawaguchi K., Meunier J. C., Takikawa S., Faulk K., Bukh J., Purcell R. H., Emerson S. U. 2009; Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles. J Viral Hepat 16:621–632 [View Article][PubMed]
     [Google Scholar]
  52. Selby M. J., Glazer E., Masiarz F., Houghton M. 1994; Complex processing and protein:protein interactions in the E2:NS2 region of HCV. Virology 204:114–122 [View Article][PubMed]
     [Google Scholar]
  53. Spadaccini R., D’Errico G., D’Alessio V., Notomista E., Bianchi A., Merola M., Picone D. 2010; Structural characterization of the transmembrane proximal region of the hepatitis C virus E1 glycoprotein. Biochim Biophys Acta 1798:344–353 [View Article][PubMed]
     [Google Scholar]
  54. 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 [View Article][PubMed]
     [Google Scholar]
  55. Vieyres G., Thomas X., Descamps V., Duverlie G., Patel A. H., Dubuisson J. 2010; Characterization of the envelope glycoproteins associated with infectious hepatitis C virus. J Virol 84:10159–10168 [View Article][PubMed]
     [Google Scholar]
  56. Wakita T., Pietschmann T., Kato T., Date T., Miyamoto M., Zhao Z., Murthy K., Habermann A., Kräusslich H. G. et al. other authors 2005; Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796 [View Article][PubMed]
     [Google Scholar]
  57. Yagnik A. T., Lahm A., Meola A., Roccasecca R. M., Ercole B. B., Nicosia A., Tramontano A. 2000; A model for the hepatitis C virus envelope glycoprotein E2. Proteins 40:355–366 [View Article][PubMed]
     [Google Scholar]
  58. 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 [View Article][PubMed]
     [Google Scholar]
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Comprehensive linker-scanning mutagenesis of the hepatitis C virus E1 and E2 envelope glycoproteins reveals new structure–function relationships
J. Gen. Virol. 92, 2249 (2011); https://doi.org/10.1099/vir.0.034314-0
/content/journal/jgv/10.1099/vir.0.034314-0
/content/journal/jgv/10.1099/vir.0.034314-0
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