1887

Abstract

We have expressed cDNA encoding the dengue virus structural proteins in by chromosomal integration of an expression cassette containing the dengue virus structural genes (CprME). The yeast recombinant E protein migrated during SDS-PAGE as a 65 kDa protein when analysed by Western blotting and radioimmunopre- cipitation, which is the expected molecular mass for correctly processed and glycosylated E protein. Treatment with endoglycosidases showed that the recombinant E protein was modified by the addition of short mannose chains. The E protein migrated with a buoyant density of 1·13 g/cm when analysed using sucrose density gradient centrifugation. Spherical structures with an average diameter of 30 nm, whose morphology resembles dengue virions, were observed in the purified fractions using transmission electron microscopy. Furthermore, the virus-like particles were immunogenic in animals and were able to induce neutralizing antibodies. This is the first report that expression of the structural genes of a flavivirus in yeast is able to generate particulate structures that resemble virions.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-78-8-1861
1997-08-01
2024-04-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/78/8/9266980.html?itemId=/content/journal/jgv/10.1099/0022-1317-78-8-1861&mimeType=html&fmt=ahah

References

  1. Chambers T. J., Hahn C. S., Galler R., Rice C. M. 1990; Flavivirus genome organisation, expression and replication. Annual Review of Microbiology 44:649–688
    [Google Scholar]
  2. Cregg J. M., Barringer K. J., Hessler A. Y., Madden K. R. 1985; Pichia pastoris as a host system for transformations. Molecular and Cellular Biology 5:3376–3385
    [Google Scholar]
  3. Cregg J. M., Tschopp J. F., Stillman C., Siegel R., Akong M., Craig W. S., Buckholtz R. G., Madden K. R., Kellaris P. A., Davis G. R., Smiley B. L., Cruze J., Torregrossa R., Velicelebi G., Thill G. P. 1987; High-level expression and efficient assembly of hepatitis B surface antigen in the methylotropic yeast Pichia pastoris. Bio/ Technology 5:479–485
    [Google Scholar]
  4. Fonsaca B. A. L., Pincus S., Shope R. E., Paoletti E., Mason P. W. 1994; Recombinant vaccinia viruses co-expressing dengue-1 glycoproteins prM and E induce neutralising antibodies in mice. Vaccine 12:279–285
    [Google Scholar]
  5. Fu J., Tan B. H., Yap E. H., Tan Y. H. 1992; Full-length cDNA sequence of dengue type 1 virus (Singapore strain S275/90). Virology 188:953–958
    [Google Scholar]
  6. Fujita H., Sumiyoshi H., Mori C., Manabe S., Takagi M., Yoshida I., Morita K., Fuka I., Fukai K., Igarashi A. 1987; Studies in the development of Japanese encephalitis vaccine: expression of virus envelope glycoprotein V3 (E) gene in yeast. Bulletin of the World Health Organization 65:303–308
    [Google Scholar]
  7. Grinna L. S., Tschopp J. F. 1989; Size distribution and general structural features of N-linked oligosaccharides from the methylotropic yeast Pichia pastoris. Yeast 5:107–115
    [Google Scholar]
  8. Guirakhoo F., Heinz F. X., Kunz C. 1989; Epitope model of tick- borne encephalitis virus envelope glycoprotein E : analysis of structural properties, role of carbohydrate side chain and conformational changes at acidic pH. Virology 169:90–99
    [Google Scholar]
  9. Kitano K., Nakao H., Itoh Y., Fujisawa Y. 1987; Recombinant hepatitis B virus surface antigen P31 accumulates as particles in Saccharomyces cerevisiae. Bio/Technology 5:281–283
    [Google Scholar]
  10. Kobayashi M., Asano T., Utsunomiya U., Itoh Y., Fujisawa Y., Nishimura O., Kato K., Kakinuma A. 1988; Recombinant hepatitis B virus surface antigen carrying the pre-S2 region derived from yeast: purification and characterisation. Journal of Biotechnology 8:1–22
    [Google Scholar]
  11. Konishi E., Pincus S., Fonseca B. A. L., Shope R. E., Paoletti E., Mason P. W. 1991; Comparison of protective immunity elicited by recombinant vaccinia viruses that synthesize E or NS1 of Japanese encephalitis virus. Virology 185:401–410
    [Google Scholar]
  12. Konishi E., Pincus S., Paoletti E., Shope R. E., Burrage T., Mason P. W. 1992; Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection. Virology 188:714–720
    [Google Scholar]
  13. Kuroda S., Otaka S., Miyazaki T., Nakao M., Fujisawa Y. 1992; Hepatitis B virus envelope L protein particles. Synthesis and assembly in Saccharomyces cerevisiae, purification and characterisation. Journal of Biological Chemistry 267:1953–1961
    [Google Scholar]
  14. Mason P. W., Pincus S., Fournier M. J., Mason T. L., Shope R. E., Paoletti E. 1991; Japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal JEV infection. Virology 180:294–305
    [Google Scholar]
  15. Murphy F. A. 1980; Togavirus morphology and morphogenesis. In The Togaviruses: Biology, Structure, Replication, pp 214–316 Schlesinger R. W. Edited by New York: Academic Press;
    [Google Scholar]
  16. Pincus S., Mason P. W., Konishi E., Fonseca B. A. L., Shope R. E., Rice C. M., Paoletti E. 1992; Recombinant vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis. Virology 187:290–297
    [Google Scholar]
  17. Schalich J., Allison S. L., Stiansny K., Mandl C. W., Kunz C., Heinz F. X. 1996; Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions. Journal of Virology 70:4549–4557
    [Google Scholar]
  18. Scorer C. A., Buckholz R. G., Clare J. J., Romanos M. A. 1993; The intracellular production and secretion of HIV-1 envelope protein in the methylotropic yeast Pichia pastoris. Gene 136:111–119
    [Google Scholar]
  19. Trimble R. B., Atkinson P. H., Tschopp J. F., Townsend R. R., Maley F. 1991; Structure of oligosaccharides on Saccharomyces SU2 invertase secreted by the methylotropic yeast Pichia pastoris. Journal of Biological Chemistry 266:22807–22817
    [Google Scholar]
  20. Winkler G., Heinz F. X., Kunz C. 1987; Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structure. Virology 159: 237–243
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-78-8-1861
Loading
/content/journal/jgv/10.1099/0022-1317-78-8-1861
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