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.

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2001-09-01
2024-04-27
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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]
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