1887

Abstract

The non-structural glycoprotein NS1 of dengue virus type 2 contains sites for -linked glycosylation at Asn-130 and Asn-207. NS1 synthesized in infected cells is glycosylated at both locations. We have now examined the dimerization and secretion of NS1 lacking one or both of these sites by transient expression of mutagenized cDNA inserted into a simian virus 40-based vector. Immunoblotting and radioimmunoprecipitation were used to detect NS1 associated with transfected cells and in the extracellular medium. Elimination of one or both glycosylation sites did not abolish dimerization and secretion of NS1. However, NS1 lacking Asn-207 showed reduced dimer stability and secretion. Treatment of secreted NS1 with endoglycosidase H demonstrated that complex glycans were attached at Asn-130 and high-mannose glycans at Asn-207.

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1994-05-01
2024-12-14
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References

  1. Cardiff R. D., Lund J. K. 1976; Distribution of dengue-2 antigens by electron immunocytochemistry. Infection and Immunity 13:1699–1709
    [Google Scholar]
  2. Chambers T. J., Hahn C. S., Galler R., Rice C. M. 1990; Flavivirus genome organization, expression, and replication. Annual Review of Microbiology 44:649–688
    [Google Scholar]
  3. Cunningham B. C., Wells J. A. 1989; High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning muta-genesis. Science 244:1081–1085
    [Google Scholar]
  4. Desprès P., Girard M., Bouloy M. 1991; Characterization of yellow fever virus proteins E and NS1 expressed in Vero and Spodoptera frugiperda cells. Journal of General Virology 72:1331–1342
    [Google Scholar]
  5. Doms R. W., Lamb R. A., Rose J. K., Helenius A. 1993; Folding and assembly of viral membrane proteins. Virology 193:545–562
    [Google Scholar]
  6. Flamand M., Deubel V., Girard M. 1992; Expression and secretion of Japanese encephalitis virus nonstructural protein NS1 by insect cells using a recombinant baculovirus. Virology 191:826–836
    [Google Scholar]
  7. Hahn Y. S., Galler R., Hunkapiller T., Dalrymple J. M., Strauss J. H., Strauss E. G. 1988; Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses. Virology 162:167–180
    [Google Scholar]
  8. Irie K., Mohan P. M., Sasaguri Y., Putnak R., Padmanabhan R. 1989; Sequence analysis of cloned dengue virus type 2 genome (New Guinea-C strain). Gene 75:197–211
    [Google Scholar]
  9. Jacobs S. C., Stephenson J. R., Wilkinson G. W. G. 1992; High- level expression of the tick-borne encephalitis virus NS1 protein by using an adenovirus-based vector: protection elicited in a murine model. Journal of Virology 66:2086–2095
    [Google Scholar]
  10. Kunkel T. A., Roberts J. D., Zakour R. A. 1987; Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymology 154:367–382
    [Google Scholar]
  11. Mahoney W. C., Duskin D. 1979; Biological activities of the two major components of tunicamycin. Journal of Biological Chemistry 254:6572–6576
    [Google Scholar]
  12. Mandl C. W., Heinz F. X., Stockl E., Kunz C. 1989; Genome sequence of tick-borne encephalitis virus (western subtype) and comparative analysis of nonstructural proteins with other flavi-viruses. Virology 173:291–301
    [Google Scholar]
  13. Mason P. W. 1989; Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells. Virology 169:354–364
    [Google Scholar]
  14. Olden K., Parent J. B., White S. L. 1982; Carbohydrate moieties of glycoproteins. A re-evaluation of their function. Biochimica et biophysica acta 650:209–232
    [Google Scholar]
  15. Pletnev A. G., Bray M., Lai C. -J. 1993; Chimeric tick-borne encephalitis and dengue type 4 viruses: effects of mutations on neurovirulence in mice. Journal of Virology 67:4956–4963
    [Google Scholar]
  16. Post P. R., Carvalho R., Galler R. 1990; Glycosylation and secretion of yellow fever virus nonstructural protein NS1. Virus Research 18:291–302
    [Google Scholar]
  17. Pryor M. J., Wright P. J. 1993; The effects of site-directed mutagenesis on the dimerization and secretion of the NS1 protein specified by dengue virus. Virology 194:769–780
    [Google Scholar]
  18. Rice C. M., Strauss E. G., Strauss J. H. 1986; Structure of the flavivirus genome. In The Togaviridae and Flaviviridae pp. 279–326 Schlesinger S., Schlesinger M. J. Edited by New York: Plenum Press;
    [Google Scholar]
  19. Smith G. W., Wright P. J. 1985; Synthesis of proteins and glycoproteins in dengue type 2 virus-infected Vero and Aedes albopictus cells. Journal of General Virology 66:559–571
    [Google Scholar]
  20. Stohlman S. A., Wisseman C. L., Eylar O. R., Silverman D. J. 1975; Dengue virus-induced modifications of host cell membranes. Journal of Virology 16:1017–1026
    [Google Scholar]
  21. Westaway E. G., Goodman M. R. 1987; Variation in distribution of the three flavivirus-specific glycoproteins detected by immuno-fluorescence in infected Vero cells. Archives of Virology 94:215–228
    [Google Scholar]
  22. Winkler G., Randolph V. B., Cleaves G. R., Ryan T. E., Stollar V. 1988; Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer. Virology 162:187–196
    [Google Scholar]
  23. Winkler G., Maxwell S. E., Ruemmler C., Stollar V. 1989; Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization. Virology 171:302–305
    [Google Scholar]
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