1887

Journal of General Virology header logo

Volume 81, Issue 12

Construction and characterization of chimeric hepatitis C virus E2 glycoproteins: analysis of regions critical for glycoprotein aggregation and CD81 binding Free

Abstract

We compared the ability of two closely related truncated E2 glycoproteins (E2) derived from hepatitis C virus (HCV) genotype 1a strains Glasgow (Gla) and H77c to bind a panel of conformation-dependent monoclonal antibodies (MAbs) and CD81. In contrast to H77c, Gla E2 formed disulfide-linked high molecular mass aggregates and failed to react with conformation-dependent MAbs and CD81. To delineate amino acid (aa) regions associated with protein aggregation and CD81 binding, several Gla–H77c E2 chimeric glycoproteins were constructed. Chimeras C1, C2 and C6, carrying aa 525–660 of Gla E2, produced disulfide-linked aggregates and failed to bind CD81 and conformation-dependent MAbs, suggesting that amino acids within this region are responsible for protein misfolding. The presence of Gla hypervariable region 1 (aa 384–406) on H77 E2, chimera C4, had no effect on protein folding or CD81 binding. Chimeras C3 and C5, carrying aa 384–524 or 407–524 of Gla E2, respectively, were recognized by conformation-dependent MAbs and yet failed to bind CD81, suggesting that amino acids in region 407–524 are important in modulating CD81 interaction without affecting antigen folding. Comparison of Gla and H77c E2 aa sequences with those of genotype 1a and divergent genotypes identified a number of variant amino acids, including two putative -linked glycosylation sites at positions 476 and 532. However, introduction of G476N–G478S and/or D532N in Gla E2 had no effect on antigenicity or aggregation.

  • Received:
  • Accepted:
  • Published Online:
© Microbiology Society
Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-81-12-2873
2000-12-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/81/12/0812873a.html?itemId=/content/journal/jgv/10.1099/0022-1317-81-12-2873&mimeType=html&fmt=ahah

References

  1. Agnello V., Abel G., Elfahal M., Knight G. B., Zhang Q. X. 1999; Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proceedings of the National Academy of Sciences, USA 96:12766–12771
     [Google Scholar]
  2. Blanchet C., Combet C., Geourjon C., Deleage G. 2000; MPSA: Integrated system for multiple protein sequence analysis with client/server capabilities. Bioinformatics 16:286–288
     [Google Scholar]
  3. Chamberlain R. W., Adams N. J., Taylor L. A., Simmonds P., Elliott R. M. 1997; The complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in South Africa. Biochemical and Biophysical Research Communications 236:44–49
     [Google Scholar]
  4. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. 1989; Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral-hepatitis genome. Science 244:359–362
     [Google Scholar]
  5. Choukhi A., Ung S., Wychowski C., Dubuisson J. 1998; Involvement of endoplasmic reticulum chaperones in the folding of hepatitis C virus glycoproteins. Journal of Virology 72:3851–3858
     [Google Scholar]
  6. Clarke B. 1997; Molecular virology of hepatitis C virus. Journal of General Virology 78:2397–2410
     [Google Scholar]
  7. 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. Journal of Virology 72:2183–2191
     [Google Scholar]
  8. Combet C., Blanchet C., Geourjon C., Deleage G. 2000; NPS@: network protein sequence analysis. Trends in Biochemical Sciences 25:147–150
     [Google Scholar]
  9. Davison A. J., Moss B. 1990; New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high level expression of foreign proteins. Nucleic Acids Research 18:4285–4286
     [Google Scholar]
  10. 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. Journal of Virology 71:697–704
     [Google Scholar]
  11. Dubuisson J., Rice C. M. 1996; Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin. Journal of Virology 70:778–786
     [Google Scholar]
  12. 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. Journal of Virology 68:6147–6160
     [Google Scholar]
  13. Farci P., Shimoda A., Wong D., Cabezon T., DeGioannis D., Strazzera A., Shimizu Y., Shapiro M., Alter H. J., Purcell R. H. 1996; Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proceedings of the National Academy of Sciences USA: 93:15394–15399
     [Google Scholar]
  14. Flint M., McKeating J. A. 2000; The role of hepatitis C virus glycoproteins in infection. Medical Virology (in press)
    [Google Scholar]
  15. Flint M., Maidens C., Loomis-Price L. D., Shotton C., Dubuisson J., Monk P., Higginbottom A., Levy S., McKeating J. A. 1999; Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81. Journal of Virology 73:6235–6244
     [Google Scholar]
  16. Flint M., Dubuisson J., Maidens C., Harrop R., Guile G. R., Borrow P., McKeating J. A. 2000; Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein. Journal of Virology 74:702–709
     [Google Scholar]
  17. Gahmberg C. G., Tolvanen M. 1996; Why mammalian cell surface proteins are glycoproteins. Trends in Biochemical Sciences 21:308–311 erratum 21, 491
    [Google Scholar]
  18. Habersetzer F., Fournillier A., Dubuisson J., Rosa D., Abrignani S., Wychowski C., Nakano I., Trepo C., Desgranges C., Inchauspe G. 1998; Characterization of human monoclonal antibodies specific to the hepatitis C virus glycoprotein E2 with in vitro binding neutralization properties. Virology 249:32–41
     [Google Scholar]
  19. Higginbottom A., Quinn E. R., Kuo C.-C., Flint M., Wilson L., Pessi A., Nicosia A., Monk P., McKeating J. A., Levy S. 2000; Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2. Journal of Virology 74:3642–3649
     [Google Scholar]
  20. Houghton M. 1996; Hepatitis C viruses. In Fields Virology. , 3rd edn. pp 1035–1058 Edited by Fields B. N., Knipe D. M., Howley P. M. Philadelphia: Lippincott–Raven;
  21. Kato N., Ootsuyama Y., Tanaka T., Nakagawa M., Nakazawa T., Muraiso K., Ohkoshi S., Hijikata M., Shimotohno K. 1992; Marked sequence diversity in the putative envelope proteins of hepatitis C viruses. Virus Research 22:107–123
     [Google Scholar]
  22. Kolykhalov A. A., Feinstone S. M., Rice C. M. 1996; Identification of a highly conserved sequence element at the 3′ terminus of hepatitis C virus genome RNA. Journal of Virology 70:3363–3371
     [Google Scholar]
  23. Lavanchy D., Purcell R., Hollinger F. B., Howard C., Alberti A., Kew M., Dusheiko G., Alter M., Ayoola E., Beutels P., Bloomer R., Ferret B., Decker R., Esteban R., Fay O., Fields H., Fuller E. C., Grob P., Houghton M., Leung N., Locarnini S. A., Margolis H., Meheus A., Miyamura T., Mohamed M. K., Tandon B., Thomas D., Head H. T., Toukan A. U., VanDamme P., Zanetti A., Arthur R., Couper M., Damelio R., Emmanuel J. C., Esteves K., Gavinio P., Griffiths E., Hallaj Z., Heuck C. C., Heymann D. L., Holck S. E., Kane M., Martinez L. J., Meslin F., Mochny I. S., Ndikuyeze A., Padilla A. M., Rodier G. R. M., Roure C., Savage F., Vercauteren G. 1999; Global surveillance and control of hepatitis C. Journal of Viral Hepatitis 6:35–47
     [Google Scholar]
  24. Levy S., Todd S. C., Maecker H. T. 1998; CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system. Annual Review of Immunology 16:89–109
     [Google Scholar]
  25. Liberman E., Fong Y. L., Selby M. J., Choo Q. L., Cousens L., Houghton M., Yen T. S. B. 1999; Activation of the grp78 and grp94 promoters by hepatitis C virus E2 envelope protein. Journal of Virology 73:3718–3722
     [Google Scholar]
  26. Matsuura Y., Suzuki T., Suzuki R., Sato M., Aizaki H., Saito I., Miyamura T. 1994; Processing of E1 and E2 glycoproteins of hepatitis C virus expressed in mammalian and insect cells. Virology 205:141–150
     [Google Scholar]
  27. 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. Journal of General Virology 78:2299–2306
     [Google Scholar]
  28. Mizushima H., Hijikata M., Asabe S., Hirota M., Kimura K., Shimotohno K. 1994; Two hepatitis C virus glycoprotein E2 products with different C termini. Journal of Virology 68:6215–6222
     [Google Scholar]
  29. Ogata N., Alter H. J., Miller R. H., Purcell R. H. 1991; Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proceedings of the National Academy of Sciences, USA 88:3392–3396
     [Google Scholar]
  30. Patel J., Patel A. H., McLauchlan J. 1999; Covalent interactions are not required to permit or stabilize the non-covalent association of hepatitis C virus glycoproteins E1 and E2. Journal of General Virology 80:1681–1690
     [Google Scholar]
  31. Pearson W. R., Lipman D. J. 1988; Improved tools for biological sequence comparison. Proceedings of the National Academy of Sciences, USA 85:2444–2448
     [Google Scholar]
  32. Pileri P., Uematsu Y., Campagnoli S., Galli G., Falugi F., Petracca R., Weiner A. J., Houghton M., Rosa D., Grandi G., Abrignani S. 1998; Binding of hepatitis C virus to CD81. Science 282:938–941
     [Google Scholar]
  33. Ralston R., Thudium K., Berger K., Kuo C., Gervase B., Hall J., Selby M., Kuo G., Houghton M., Choo Q. L. 1993; Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. Journal of Virology 67:6753–6761
     [Google Scholar]
  34. Robertson B., Myers G., Howard C., Brettin T., Bukh J., Gaschen B., Gojobori T., Maertens G., Mizokami M., Nainan O., Netesov S., Nishioka K., Shini T., Simmonds P., Smith D., Stuyver L., Weiner A. 1998; Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. Archives of Virology 143:2493–2503
     [Google Scholar]
  35. Rosa D., Campagnoli S., Moretto C., Guenzi E., Cousens L., Chin M., Dong C., Weiner A. J., Lau J. Y. N., Choo Q. L., Chien D., Pileri P., Houghton M., Abrignani S. 1996; A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proceedings of the National Academy of Sciences, USA 93:1759–1763
     [Google Scholar]
  36. Rose J. K., Buonocore L., Whitt M. A. 1991; A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques 10:520–525
     [Google Scholar]
  37. Ryan M. D., Monaghan S., Flint M. 1998; Virus-encoded proteinases of the Flaviviridae. Journal of General Virology 79:947–959
     [Google Scholar]
  38. 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
     [Google Scholar]
  39. Shimizu Y. K., Igarashi H., Kiyohara T., Cabezon T., Farci P., Purcell R. H., Yoshikura H. 1996; A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures. Virology 223:409–412
     [Google Scholar]
  40. Simmonds P. 1995; Variability of hepatitis C virus. Hepatology 21:570–583
     [Google Scholar]
  41. Simmonds P., Alberti A., Alter H. J., Bonino F., Bradley D. W., Brechot C., Brouwer J. T., Chan S. W., Chayama K., Chen D. S., Choo Q. L., Colombo M., Cuypers H. T. M., Date T., Dusheiko G. M., Esteban J. I., Fay O., Hadziyannis S. J., Han J., Hatzakis A., Holmes E. C., Hotta H., Houghton M., Irvine B., Kohara M., Kolberg J. A., Kuo G., Lau J. Y. N., Lelie P. N., Maertens G., McOmish F., Miyamura T., Mizokami M., Nomoto A., Prince A. M., Reesink H. W., Rice C., Roggendorf M., Schalm S. W., Shikata T., Shimotohno K., Stuyver L., Trepo C., Weiner A., Yap P. L., Urdea M. S. 1994; A proposed system for the nomenclature of hepatitis C viral genotypes. Hepatology 19:1321–1324
     [Google Scholar]
  42. Spaete R. R., Alexander D., Rugroden M. E., Choo Q. L., Berger K., Crawford K., Kuo C., Leng S., Lee C., Ralston R. and others 1992; Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells. Virology 188:819–830
     [Google Scholar]
  43. Thompson J. D., Higgins D. G., Gibson T. J. 1994; CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22:4673–4680
     [Google Scholar]
  44. Weiner A. J., Brauer M. J., Rosenblatt J., Richman K. H., Tung J., Crawford K., Bonino F., Saracco G., Choo Q. L., Houghton M., Han J. H. 1991; Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and Ns1 proteins and the pestivirus envelope glycoproteins. Virology 180:842–848
     [Google Scholar]
  45. 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. Proceedings of the National Academy of Sciences, USA 94:8738–8743
     [Google Scholar]
  46. Zibert A., Schreier E., Roggendorf M. 1995; Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment. Virology 208:653–663
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-81-12-2873
Loading
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 (2000); https://doi.org/10.1099/0022-1317-81-12-2873
/content/journal/jgv/10.1099/0022-1317-81-12-2873
/content/journal/jgv/10.1099/0022-1317-81-12-2873
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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