Skip to content
1887

Abstract

The 12 cysteine residues in the flavivirus NS1 protein are strictly conserved, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. In this study, we examined the intramolecular disulfide bond arrangement of NS1 of Murray Valley encephalitis virus and elucidated three of the six cysteine-pairing arrangements. Disulfide linkages were identified by separating tryptic-digested NS1 by reverse-phase high pressure liquid chromatography and analysing the resulting peptide peaks by protein sequencing, amino acid analysis and/or electrospray mass spectrometry. The pairing arrangements between the six amino-terminal cysteines were identified as follows: Cys–Cys, Cys–Cys and Cys–Cys. Although the pairing arrangements between the six carboxy-terminal cysteines were not determined, we were able to eliminate several cysteine-pairing combinations. Furthermore, we demonstrated that all three putative -linked glycosylation sites of NS1 are utilized and that the Asn glycosylation site contains a mannose-rich glycan.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-82-9-2251
2001-09-01
2025-03-21
Loading full text...

Full text loading...

References

  1. Blitvich B. J., Scanlon D., Shiell B. J., Mackenzie J. S., Hall R. A. 1999; Identification and analysis of truncated and elongated species of the flavivirus NS1 protein. Virus Research 60:67–79
    [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. Crooks A. J., Lee J. M., Easterbrook L. M., Timofeev A. V., Stephenson J. R. 1994; The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell. Journal of General Virology 75:3453–3460
    [Google Scholar]
  4. Dalgarno L., Trent D. W., Strauss J. H., Rice C. M. 1986; Partial nucleotide sequence of the Murray Valley encephalitis virus genome: comparison of the encoded polypeptides with yellow fever virus structural and nonstructural proteins. Journal of Molecular Biology 187:309–323
    [Google Scholar]
  5. Falgout B., Markoff L. 1995; The family Flaviviridae and its diseases. In Exotic Viral Infections pp 47–66 Edited by Porterfield J. S. London: Chapman and Hall Medical;
    [Google Scholar]
  6. Flamand M., Megret F., Mathieu M., Lepault J., Rey F. A., Deubel V. 1999; Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. Journal of Virology 73:6104–6110
    [Google Scholar]
  7. Gorman J. J., Shiell B. J. 1993; Isolation of carboxyl-termini and blocked amino-termini of viral proteins by high-performance cation-exchange chromatography. Journal of Chromatography 646:193–205
    [Google Scholar]
  8. Hall R. A., Kay B. H., Burgess G. W., Clancy P., Fanning I. D. 1990; Epitope analysis of the envelope and nonstructural glycoproteins of Murray Valley encephalitis virus. Journal of General Virology 71:2923–2930
    [Google Scholar]
  9. Hall R. A., Coelen R. J., Mackenzie J. S. 1991; Immunoaffinity purification of the NS1 protein of Murray Valley encephalitis virus: selection of the appropriate ligand and optimal conditions for elution. Journal of Virological Methods 32:11–20
    [Google Scholar]
  10. Hall R. A., Brand T. N., Lobigs M., Sangster M. Y., Howard M. J., Mackenzie J. S. 1996; Protective immune responses to the E and NS1 proteins of Murray Valley encephalitis virus in hybrids of flavivirus-resistant mice. Journal of General Virology 77:1287–1294
    [Google Scholar]
  11. Khromykh A. A., Sedlak P. L., Guyatt K. J., Hall R. A., Westaway E. G. 1999; Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase. Journal of Virology 73:10272–10280
    [Google Scholar]
  12. Kornfeld R., Kornfeld S. 1985; Assembly of asparagine-linked oligosaccharides. Annual Review of Biochemistry 54:631–664
    [Google Scholar]
  13. Lindenbach B. D., Rice C. M. 1997; Trans-complementation of YF virus NS1 reveals a role in early RNA replication. Journal of Virology 71:9608–9617
    [Google Scholar]
  14. Mackenzie J. S., Lindsay M. D., Coelen R. J., Broom A. K., Hall R. A., Smith D. W. 1994; Arboviruses causing human disease in the Australasian zoogeographic region. Archives of Virology 136:447–467
    [Google Scholar]
  15. Mackenzie J. M., Jones M. K., Young P. R. 1996; Immunolocalization of the dengue viral nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 220:220–240
    [Google Scholar]
  16. Mackow E., Makino Y., Zhao B. T., Zhang Y. M., Markoff L., Buckler-White A., Guiler M., Chanock R., Lai C. J. 1987; The nucleotide sequence of dengue type 4 virus: analysis of genes coding for nonstructural proteins. Virology 159:217–228
    [Google Scholar]
  17. Mason P. W. 1989; Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells. Virology 169:354–364
    [Google Scholar]
  18. Mason P. W., McAda P. C., Dalrymple J. M., Fournier M. J., Mason T. C. 1987; Expression of Japanese encephalitis virus antigens in Escherichia coli. Virology 158:361–372
    [Google Scholar]
  19. Monath T. P., Heinz F. X. 1996; Flaviviruses. In Fields Virology pp 961–1034 Edited by Fields B. N., Knipe D. M., Howley P. M. Philadelphia: Lippincott–Raven;
    [Google Scholar]
  20. Muylaert I. A., Chambers T. J., Galler R., Rice C. M. 1996; Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein: effects on virus replication and mouse virulence. Virology 222:159–168
    [Google Scholar]
  21. Muylaert I. A., Galler R., Rice C. M. 1997; Genetic analysis of the yellow fever virus NS1 protein: identification of a temperature sensitive mutation which blocks RNA accumulation. Journal of Virology 71:291–298
    [Google Scholar]
  22. Nestorowicz A., Chambers T. J., Rice C. M. 1994; Mutagenesis of the yellow fever virus NS2A/2B cleavage site: effects on proteolytic processing, viral replication and evidence for alternative processing of the NS2A protein. Virology 199:114–123
    [Google Scholar]
  23. Pryor M. J., Wright P. J. 1993; The effects of site-directed mutagenesis on the dimerization and secretion of the NS1 protein specific by dengue virus. Virology 194:769–780
    [Google Scholar]
  24. Pryor M. J., Wright P. J. 1994; Glycosylation mutants of dengue virus NS1 protein. Journal of General Virology 75:1183–1187
    [Google Scholar]
  25. Rice C. M. 1996; Flaviviridae : the viruses and their replication. In Fields Virology pp 931–959 Edited by Fields B. N., Knipe D. M., Howley P. M. Philadelphia: Lippincott–Raven;
    [Google Scholar]
  26. Schlesinger J. J., Brandriss M. W., Cropp C. B., Monath T. P. 1986; Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NS1. Journal of Virology 60:1153–1155
    [Google Scholar]
  27. Schlesinger J. J., Brandriss M. W., Putnak J. R., Walsh E. E. 1990; Cell surface expression of yellow fever virus non-structural glycoprotein NS1: consequences of interaction with antibody. Journal of General Virology 71:593–599
    [Google Scholar]
  28. Stadler K., Allison S. L., Schalich J., Heinz F. X. 1997; Proteolytic activation of tick-borne encephalitis virus by furin. Journal of Virology 71:8475–8481
    [Google Scholar]
  29. Sumiyoshi H., Mori C., Fuke I., Morita K., Kuhara S., Kondou J., Kikuchi Y., Nagamatu H., Igarashi A. 1987; Complete nucleotide sequence of the Japanese encephalitis virus genome RNA. Virology 161:497–510
    [Google Scholar]
  30. Timofeev A. V., Ozherelkov S. V., Pronin A. V., Deeva A. V., Karganova G. G., Elbert L. B., Stephenson J. R. 1998; Immunological basis for protection in a murine model of tick-borne encephalitis by a recombinant adenovirus carrying the gene encoding the NS1 non-structural protein. Journal of General Virology 79:689–695
    [Google Scholar]
  31. 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]
  32. 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]
  33. Young P. R., Falconar A. K. I. 1989; Nonstructural proteins as virus vaccines. Arbovirus Research in Australia 5:62–67
    [Google Scholar]
/content/journal/jgv/10.1099/0022-1317-82-9-2251
Loading
/content/journal/jgv/10.1099/0022-1317-82-9-2251
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