1887

Abstract

Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, which are thought to locate to the envelope of virus particles. These proteins form two complexes in tissue culture systems, a high molecular mass aggregate that contains intermolecular covalent bonds and a native complex in which E1 and E2 associate by non-covalent interactions. The contribution of either complex to the structures of the proteins on virus particles is not known. Using dithiothreitol to reduce inter- and intramolecular disulphide bonds in situ, we have studied the nature of the interactions within the aggregate and the role of covalent bonds in the early stages of E1-E2 association. Results with two HCV type 1a strains, Glasgow and H77, showed that the aggregate contains not only covalent interactions but also non-covalent associations between E1 and E2. These non-covalent associations are complex since deletion mutant analysis failed to identify any single region which was required for non-covalent interaction. Complex formation by de novo synthesized proteins was not arrested under reducing conditions which prevented the production of inter- and intramolecular disulphide bonds. Moreover, a conformation-specific antibody continued to recognize the E2 protein in reduced complexes, indicating that covalent bonds do not stabilize certain structures of E2 that can interact with E1. These data suggest that disulphide bonds are not required either to allow association between the proteins or to stabilize E1-E2 complexes.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-80-7-1681
1999-07-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/80/7/0801681a.html?itemId=/content/journal/jgv/10.1099/0022-1317-80-7-1681&mimeType=html&fmt=ahah

References

  1. Braakman I., Helenius J., Helenius A. 1992; Manipulating disulphide bond formation and protein folding in the endoplasmic reticulum. EMBO Journal 11:1717–1722
    [Google Scholar]
  2. Carleton M., Brown D. T. 1996; Disulphide bridge-mediated folding of Sindbis virus glycoproteins. Journal of Virology 70:5541–5547
    [Google Scholar]
  3. Carleton M., Brown D. T. 1997; The formation of intramolecular disulphide bridges is required for induction of the Sindbis virus mutant ts23 phenotype. Journal of Virology 71:7696–7703
    [Google Scholar]
  4. Clarke B. E. 1997; Molecular virology of hepatitis C virus. Journal of General Virology 78:2397–2410
    [Google Scholar]
  5. 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]
  6. 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]
  7. Dubuisson J., Rice C. M. 1996; Hepatitis C virus glycoprotein folding: disulphide bond formation and association with calnexin. Journal of Virology 70:778–786
    [Google Scholar]
  8. 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]
  9. Fournillier-Jacob A., Cahour A., Escriou N., Girard M., Wychowski C. 1996; Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells. Journal of General Virology 77:1055–1064
    [Google Scholar]
  10. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. 1993; Expression and identification of hepatitis C virus polyprotein cleavage products. Journal of Virology 67:1385–1395
    [Google Scholar]
  11. Inchauspe G., Zebedee S., Lee D. H., Sugitani M., Nasoff M., Prince A. M. 1991; Genomic structure of the human prototype strain H of hepatitis C virus – comparison with American and Japanese isolates. Proceedings of the National Academy of Sciences, USA 88:10292–10296
    [Google Scholar]
  12. 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. Science 277:570–574
    [Google Scholar]
  13. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  14. Lee K. J., Suh Y.-A., Cho Y. G., Cho Y. S., Ha G. W., Chung K.-H., Hwang J. H., Yun Y. D., Lee D. S., Kim C. M., Sung Y.-C. 1997; Hepatitis C virus E2 protein purified from mammalian cells is frequently recognized by E2-specific antibodies in patient sera. Journal of Biological Chemistry 272:30040–30046
    [Google Scholar]
  15. Liljestrom P., Garoff H. 1991; A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology 9:1356–1361
    [Google Scholar]
  16. Lin C., Lindenbach B. D., Pragai D., McCourt D. W., Rice C. M. 1994; Processing of the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C-termini. Journal of Virology 68:5063–5073
    [Google Scholar]
  17. Matsuura Y., Suzuki T., Suzuki R., Sato M., Aizaki H., Saito I., Miyamura T. 1994; Processing of E1 and E2 glycoproteins ofhepatitis C virus expressed in mammalian and insect cells. Virology 205:141–150
    [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76:3350–3353
    [Google Scholar]
  23. Verde C., Pascale M. C., Martire G., Lotti L. V., Torrisi M. R., Helenius A., Bonatti S. 1995; Effect of ATP depletion and DTT on the transport of membrane proteins from the endoplasmic reticulum and the intermediate compartment to the Golgi complex. European Journal of Cell Biology 67:267–274
    [Google Scholar]
  24. 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]
  25. Yi M. K., Nakamoto Y., Kaneko S., Yamashita T., Murakami S. 1997; Delineation of regions important for heteromeric association of hepatitis C virus E1 and E2. Virology 231:119–129
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-80-7-1681
Loading
/content/journal/jgv/10.1099/0022-1317-80-7-1681
Loading

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