1887

Abstract

A series of recombinant baculoviruses was constructed in order to study the influence of downstream NS2A sequences on the processing of the dengue virus NS1 glycoprotein in insect cells. NS1 alone was expressed at a high level in its native dimeric form and processed efficiently through the (Sf) cell secretory pathway. Recombinant NS1 was found associated with the plasma membrane and was also secreted into the extracellular medium. Although both intra- and extracellular NS1 were processed to an endo H-resistant form in Sf cells, Triton X-114 phase separation analysis further suggested that some modifications in addition to dimerization account for the hydrophobic properties of NS1, and that -glycosylation was therefore not the only difference between the cell-associated and secreted forms. Cleavage at the NS1-NS2A junction of these recombinants demonstrated that as few as 26 amino acids from the N terminus of NS2A provide a sufficient, but not optimal, recognition sequence for a functional cleavage mediated by a protease present in Sf cells infected with recombinant nuclear polyhedrosis virus expressing NS1.

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1995-04-01
2024-04-26
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References

  1. Bordier C. 1981; Phase separation of integral membrane proteins in Triton X114 solution. Journal of Biological Chemistry 256:1604–1607
    [Google Scholar]
  2. Butters T. D., Hughes R. C. 1981; Isolation and characterization of mosquito cell membrane glycoproteins. Biochimica et Biophysica Acta 640:655–671
    [Google Scholar]
  3. Cane P. A., Gould E. A. 1988; Reduction of yellow fever virus mouse neurovirulence by immunization with a bacterially synthesized non-structural protein (NS1) fragment. Journal of General Virology 69:1241–1246
    [Google Scholar]
  4. Chambers T. J., McCourt D. W., Rice C. M. 1989; Yellow fever virus proteins NS2A, NS2B and NS4B: identification and partial N-terminal amino acid sequence analysis. Virology 169:100–109
    [Google Scholar]
  5. Chambers T. J., McCourt D. W., Rice C. M. 1990; Production of yellow fever virus proteins in infected cells: identification of discrete polyprotein species and analysis of cleavage kinetics using region specific polyclonal antisera. Virology 177:159–174
    [Google Scholar]
  6. Crooks A. J., Lee J. M., Dowsett A. B., Stephenson J. R. 1990; Purification and analysis of infectious virions and native nonstructural antigens from cells infected with tick-borne encephalitis virus. Journal of Chromatography 502:59–68
    [Google Scholar]
  7. Després 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]
  8. Falconar A. K. I., Young P. R. 1990; Immunoaffinity purification of native dimer forms of the flavivirus non-tructural glycoprotein, NS1. Journal of Virological Methods 30:323–332
    [Google Scholar]
  9. Falgout B., Chanock R., Lai C. J. 1989; Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2A. Journal of Virology 63:1852–1860
    [Google Scholar]
  10. Fan W., Mason P. W. 1990; Membrane association and secretion of the Japanese encephalitis virus NS1 protein from cells expressing NS1 cDNA. Virology 177:470–476
    [Google Scholar]
  11. Flamand M., Deubel V., Girard M. 1992; Expression and secretion of Japanese encephalitis nonstructural protein NS1 by insect cells using a recombinant baculovirus. Virology 191:826–836
    [Google Scholar]
  12. Gould E. A., Buckley A., Cammack N., Barrett A. D. T., Clegg J. C. S., Ishak R., Varma G. R. 1985; Examination of the immunological relationship between flaviviruses using yellow fever virus monoclonal antibodies. Journal of General Virology 66:1369–1382
    [Google Scholar]
  13. Gould E. A., Buckley A., Barrett A. D. T., Cammack N. 1986; Neutralizing (54K and 48K) monoclonal antibodies against structural and non-structural yellow fever virus proteins confer immunity in mice. Journal of General Virology 67:591–595
    [Google Scholar]
  14. Henchal E. A., Henchal L. S., Schlesinger J. J. 1988; Synergistic interactions of anti-NS1 antibodies protect passively immunized mice from lethal challenge with dengue 2 virus. Journal of General Virology 69:2101–2107
    [Google Scholar]
  15. Hori H., Lai C. J. 1990; Cleavage of dengue virus NS1–NS2A requires an octapeptide sequence at the C-terminus of NS1. Journal of Virology 64:4573–4577
    [Google Scholar]
  16. Jarvis D. L., Summers M. D. 1989; Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected cells. Molecular and Cellular Biology 9:214–223
    [Google Scholar]
  17. Leblois H., Young P. R. 1993; Sequence of the dengue virus type 2 (strain PR-159) NS1 gene and comparison with its vaccine derivative. Nucleic Acids Research 21:1668
    [Google Scholar]
  18. Mason P. W. 1989; Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian or mosquito cells. Virology 169:354–364
    [Google Scholar]
  19. Muylaert I. R., Galer R., Rice C. M. 1994; Genetic analysis of the NS1 protein of yellow fever virus. American Society for Virology 13th Annual Meeting, July 9–13
    [Google Scholar]
  20. Parrish C. R., Woo W. S., Wright P. J. 1991; Expression of the NS1 gene of dengue virus type 2 using vaccinia virus. Archives of Virology 117:279–286
    [Google Scholar]
  21. Pryor M., 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]
  22. Putnak J. R., Charles P. C., Padmanabhan R., Irie K., Hoke C. H., Burke D. S. 1988; Functional and antigenic domains on the dengue virus nonstructural glycoprotein NS1. Virology 163:93–103
    [Google Scholar]
  23. Rice C. M., Srauss E. G., Stauss J. H. 1986; Structure of the flavivirus genome. In The Togaviridae and Flaviviridae pp 279–326 Edited by Schlesinger S., Schlesinger M. J. New York: Plenum Press;
    [Google Scholar]
  24. Schlesinger J. J., Brandriss M. W., Walsh E. E. 1987; Protection of mice against dengue 2 virus encephalitis by immunization with the dengue 2 virus nonstructural glycoprotein NS1. Journal of General Virology 68:853–857
    [Google Scholar]
  25. Smith G. W., Wright P. J. 1985; Synthesis of proteins and glycoproteins in dengue 2 virus infected Vero and Aedes albopictus cells. Journal of General Virology 66:559–571
    [Google Scholar]
  26. Speight G., Coia G., Parker M. D., Westaway E. G. 1988; Gene mapping and positive identification of the non-structural proteins NS2A, NS2B, NS3, NS4B and NS5 of the flavivirus Kunjin and their cleavage sites. Journal of General Virology 69:23–34
    [Google Scholar]
  27. Tarentino A. L., Maley F. 1975; A comparison of the substrate specificities of cndo-β-N-acetylglucosaminidases from Streptomyces griseus and Diplococcus pneumoniae . Biochemical and Biophysical Research Communications 67:455–462
    [Google Scholar]
  28. von Heinje G. 1983; Patterns of amino acids near signal sequence cleavage sites. European Journal of Biochemistry 133:17–21
    [Google Scholar]
  29. Young P. R., Falconar A. K. I. 1990; Non–structural proteins as viral vaccines. Proceedings of the 5th Symposium on Arbovirus Research in Australia pp 62–67
    [Google Scholar]
  30. Young P. R., Bletchy C., Mackenzie J. 1993; Antigenic and structural analysis of the dengue virus glycoprotein, NS1. Proceedings of the 6th Symposium on Arbovirus Research in Australia pp 138–142
    [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 non-structural 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 vims non-structural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane– associated after dimerization. Virology 171:302–305
    [Google Scholar]
  33. Wright P. J., Cauchi M. R., Ng M. L. 1989; Definition of the carboxy termini of the three glycoproteins specified by dengue vims type 2. Virology 171:61–67
    [Google Scholar]
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