1887

Abstract

Hepatitis C virus (HCV) E1 and E2 glycoproteins assemble intracellularly to form a non-covalently linked heterodimer, which is retained in the endoplasmic reticulum (ER). To study the subcellular localization of E2 in live cells, the enhanced green fluorescent protein (EGFP) was fused to the N terminus of E2. Using fluorescence and confocal microscopy, we have confirmed that E2 is located in the ER, where budding of HCV virions is thought to occur. Immunoprecipitation experiments using a conformation-sensitive antibody and a GST pull-down assay showed that fusion of EGFP to E2 interferes neither with its heterodimeric assembly with E1, nor with proper folding of the ectodomain, nor with the capacity of E2 to interact with human CD81, indicating that the EGFP–E2 fusion protein is functional. As a tool to study binding of E2 to target cells, we also described the expression of an EGFP–E2 fusion protein at the cell surface.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.18927-0
2003-03-01
2020-08-10
Loading full text...

Full text loading...

/deliver/fulltext/jgv/84/3/vir840561.html?itemId=/content/journal/jgv/10.1099/vir.0.18927-0&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. Proc Natl Acad Sci U S A96:12766–12771
    [Google Scholar]
  2. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C.. 1994; Green fluorescent protein as a marker for gene expression. Science263:802–805
    [Google Scholar]
  3. Chen Y., Maguire T., Hileman R. E., Fromm J. R., Esko J. D., Linhardt R. J., Marks R. M.. 1997; Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nature Med3:866–871
    [Google Scholar]
  4. Choukhi A., Ung S., Wychowski C., Dubuisson J.. 1998; Involvement of endoplasmic reticulum chaperones in the folding of hepatitis C virus glycoproteins. J Virol72:3851–3858
    [Google Scholar]
  5. Cocquerel L., Meunier J.-C., Pillez A., Wychowski C., Dubuisson J.. 1998; A retention signal necessary and sufficient for endoplasmic reticulum localisation maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J Virol72:2183–2191
    [Google Scholar]
  6. Cocquerel L., Duvet S., Meunier J. C., Pillez A., Cacan R., Wychowski C., Dubuisson J.. 1999; The transmembrane domain of hepatitis C virus is a signal for static retention in the endoplasmic reticulum. J Virol73:2641–2649
    [Google Scholar]
  7. Cocquerel L., Wychowski C., Minner F., Penin F., Dubuisson J.. 2000; Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a key role in the processing, subcellular localisation and assembly of these envelope proteins. J Virol74:3623–3633
    [Google Scholar]
  8. Cocquerel L., Meunier J.-C., Op de Beeck A., Bonte D., Wychowski C., Dubuisson J.. 2001; Coexpression of hepatitis C virus envelope proteins E1 and E2 in cis improves the stability of membrane insertion of E2. J Gen Virol82:1629–1635
    [Google Scholar]
  9. 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 Virol71:697–704
    [Google Scholar]
  10. Dietzschold B., Cox J. H., Schneider L. G., Wiktor T. J., Koprowski H.. 1978; Isolation and purification of a polymeric form of the glycoprotein of rabies virus. J Gen Virol40:131–139
    [Google Scholar]
  11. Dubuisson J.. 2000; Folding, assembly and subcellular localisation of hepatitis C virus glycoproteins. Curr Top Microbiol Immunol242:135–148
    [Google Scholar]
  12. Dubuisson J., Rice C. M.. 1996; Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin. J Virol70:778–786
    [Google Scholar]
  13. Dubuisson J., Hsu H. H., Cheung R. C., Greenberg H. B., Russell D. G., Rice C. M.. 1994; Formation and intracellular localisation of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. J Virol68:6147–6160
    [Google Scholar]
  14. Duvet S., Cocquerel L., Pillez A., Cacan R., Verbert A., Moradpour D., Wychowski C., Dubuisson J.. 1998; Hepatitis C virus glycoprotein complex localisation in the endoplasmic reticulum involves a determinant for retention and not retrieval. J Biol Chem273:32088–32095
    [Google Scholar]
  15. Flint M., McKeating J. A.. 1999; The C-terminal region of the hepatitis C virus E1 glycoprotein confers localisation within the endoplasmic reticulum. J Gen Virol80:1943–1947
    [Google Scholar]
  16. Flint M., Thomas J. M., Maidens C. M., Shotton C., Levy S., Barclay W. S., McKeating J. A.. 1999; Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol73:6782–6790
    [Google Scholar]
  17. 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. J Virol74:702–709
    [Google Scholar]
  18. Forns X., Allander T., Rohwer-Nutter P., Burkh J.. 2000; Characterization of modified hepatitis C virus E2 proteins expressed on the cell surface. Virology274:75–85
    [Google Scholar]
  19. Francki R. I. B., Fauquet C. M., Knudson D. L., Brown F.. 1991; Classification and nomenclature of viruses. Fifth Report of the International Commmittee on Taxonomy of Viruses. Arch Virol Supplementum2223
    [Google Scholar]
  20. Heile J. M., Fong Y. L., Rosa D.. 17 other authors 2000; Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates. J Virol74:6885–6892
    [Google Scholar]
  21. Higginbottom A., Quinn E. R., Kuo C. C.. 7 other authors 2000; Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2. J Virol74:3642–3649
    [Google Scholar]
  22. Kieny M.-P., Lathe R., Drillien R., Spenher D., Skory S., Schmitt D., Wiktor T., Koprowski H., Lecocq J.-P.. 1984; Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature312:163–166
    [Google Scholar]
  23. Kolykhalov A. A., Agapov E. V., Blight K. J., Mihalik K., Feinstone S. M., Rice C. M.. 1997; Transmission of Hepatitis C by intrahepatic inoculation with transcribed RNA. Science277:570–574
    [Google Scholar]
  24. Mackenzie J. M., Westaway E. G.. 2001; Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J Virol75:10787–10799
    [Google Scholar]
  25. Meola A., Sbardellati A., Bruni Ercole B.. 9 other authors 2000; Binding of hepatitis C virus E2 glycoprotein to CD81 does not correlate with species permissiveness to infection. J Virol74:5933–5938
    [Google Scholar]
  26. 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 Virol78:2299–2306
    [Google Scholar]
  27. Ogawa H., Inouye S., Tsuji F. I., Yasuda K., Umesono K.. 1995; Localisation, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells. Proc Natl Acad Sci U S A9211899–11903
    [Google Scholar]
  28. Op De Beeck A., Montserret R., Duvet S., Cocquerel L., Cacan R., Barberot B., Le Maire M., Penin F., Dubuisson J.. 2000; Role of the transmembrane domains of hepatitis C virus envelope proteins E1 and E2 in the assembly of the noncovalent E1E2 heterodimer. J Biol Chem275:31428–31437
    [Google Scholar]
  29. Op De Beeck A., Cocquerel L., Dubuisson J.. 2001; Biogenesis of hepatitis C virus envelope glycoproteins. J Gen Virol82:2589–2595
    [Google Scholar]
  30. 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. Virology279:58–68
    [Google Scholar]
  31. Petracca R., Falugi F., Galli G.. 9 other authors 2000; Structure–function analysis of hepatitis C virus envelope-CD81 binding. J Virol74:2873–2883
    [Google Scholar]
  32. Pileri P., Uematsu Y., Campagnoli S.. 8 other authors 1998; Binding of hepatitis C virus to CD81. Science282:938–941
    [Google Scholar]
  33. Reed K. E., Rice C. M.. 2000; Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol242:55–84
    [Google Scholar]
  34. Scarselli E., Ansuini H., Cerino R.. 7 other authors 2002; The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J21:5017–5025
    [Google Scholar]
  35. Takikawa S., Ishii K., Aizaki H., Suzuki T., Asakura H., Matsuura Y., Miyamura T.. 2000; Cell fusion activity of hepatitis C virus envelope proteins. J Virol74:5066–5074
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.18927-0
Loading
/content/journal/jgv/10.1099/vir.0.18927-0
Loading

Data & Media loading...

Most cited this month 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