1887

Abstract

The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome, (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties, (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface, (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not oligomerize and were retained in the endoplasmic reticulum. In conclusion, cysteine residues 287, 300, 381, 394, 494, 579 and 583 appear to play a particularly critical role in the antigenic structure and processing of the H molecules and they probably participate in the inter- or intramolecular disulphide bonding.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-75-9-2173
1994-09-01
2022-05-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/75/9/JV0750092173.html?itemId=/content/journal/jgv/10.1099/0022-1317-75-9-2173&mimeType=html&fmt=ahah

References

  1. Alkhatib G., Briedis D. J. 1986; The predicted primary structure of the measles virus hemagglutinin. Virology 150:479–490
    [Google Scholar]
  2. Curran M. D., Clarke D. K., Rima B. K. 1991; The nucleotide sequence of the gene encoding the attachment protein H of canine distemper virus. Journal of General Virology 72:443–447
    [Google Scholar]
  3. Curran M. D., O’Loan D., Kennedy S., Rima B. K. 1992; Molecular characterization of phocine distemper virus: gene order and sequence of the gene encoding the attachment (H) protein. Journal of General Virology 73:1189–1194
    [Google Scholar]
  4. Doms R. W., Helenius A. 1986; Quaternary structure of influenza virus hemagglutinin after acid treatment. Journal of Virology 60:833–839
    [Google Scholar]
  5. Drillien R., Spehner T., Kirn A., Giraudon P., Buckland R., Wild T. F., Lecocq J. P. 1988; Protection of mice from fatal measles encephalitis by vaccination with vaccinia virus recombinants encoding either the hemagglutinin or the fusion protein. Proceedings of the National Academy of Sciences U.S.A: 851252–1256
    [Google Scholar]
  6. Earl P. L., Doms R. W., Moss B. 1990; Oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein. Proceedings of the National Academy of Sciences U.S.A: 87648–652
    [Google Scholar]
  7. Eschle D. 1988 Cloning and reconstruction of the entire measles virus genome Diploma thesis University of Zurich:
    [Google Scholar]
  8. Fuerst T. R., Niles E. G., Studier F. W., Moss B. 1986; Eukaryotic transient-expressing system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proceedings of the National Academy of Sciences U.S.A: 838122–8126
    [Google Scholar]
  9. Garoff H. 1985; Using recombinant DNA techniques to study protein targeting in the eucaryotic ceil. Annual Review of Cell Biology 1:403–445
    [Google Scholar]
  10. Gerald C., Buckland R., Barker R., Freeman G., Wild T. F. 1986; Measles virus haemagglutinin gene: cloning, complete nucleotide sequence analysis and expression in COS cells. Journal of General Virology 67:2695–2703
    [Google Scholar]
  11. Gething M. -J., Bye J., Skehel J., Waterfield M. 1980; Cloning and DNA sequence of double-stranded copies of haemagglutinin gene from H2 and H3 strains elucidates antigenic shift and drift in human influenza virus. Nature; London: 287301–307
    [Google Scholar]
  12. Giese N. A., Robbins K. C., Aaronson S. A. 1987; The role of individual cysteine residues in the structure and function of the V-sis gene product. Science 236:1315–1318
    [Google Scholar]
  13. Glocker M. O., Arbogast B., Schreurs J., Deinzer M. L. 1993; Assignment of the inter- and intramolecular disulphide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry. Biochemistry 32:482–488
    [Google Scholar]
  14. Haniu M., Rohde M. F., Kenney W. C. 1993; Disulphide bonds in recombinant platelet-derived growth factor BB dimer: characterization of intermolecular and intramolecular disulphide linkages. Biochemistry 32:2431–2437
    [Google Scholar]
  15. Hu A., Sheshberadaran H., Norrby E., Kövamees J. 1993; Molecular characterization of epitopes on the measles virus hemagglutinin. Virology 192:351–354
    [Google Scholar]
  16. Hu A., Kövamees J., Norrby E. 1994a; Intracellular processing and antigenic maturation of the measles virus hemagglutinin protein. Archives of Virology in press
    [Google Scholar]
  17. Hu A., Cattaneo R., Schwartz S., Norrby E. 1994b; Role of A-linked oligosaccharide chains in the processing and antigenicity of measles virus haemagglutinin protein. Journal of General Virology 75:1043–1052
    [Google Scholar]
  18. Kövamees J., Blixenkrone-Möller M., Norrby E. 1991a; The nucleotide and predicted amino acid sequence of the attachment protein of canine distemper virus. Virus Research 19:223–234
    [Google Scholar]
  19. Kövamees J., Blixenkrone-Möller M., Sharma B., Örvell C., Norrby E. 1991b; The nucleotide sequence and deduced amino acid composition of the haemagglutinin and fusion proteins of the morbillivirus phocid distemper virus. Journal of General Virology 72:2959–2966
    [Google Scholar]
  20. Leonard C. K., Spellman M. W., Riddle L., Harries R. J., Thomas J. N., Gregory T. J. 1990; Assignment of intrachain disulphide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gpl20) expressed in Chinese hamster ovary cells. Journal of Biological Chemistry 265:10373–10382
    [Google Scholar]
  21. Long D., Wilcox W. C., William R. A., Cohen G. H., Eisenberg R. J. 1992; Disulphide bond structure of glycoprotein D of herpes simplex virus type 1 and 2. Journal of Virology 66:6668–6685
    [Google Scholar]
  22. Mackett M., Smith G. L., Moss B. 1985; The construction and characterization of vaccinia virus recombinants expressing foreign genes. In DNA Cloning 2 pp 191–211 Rickwood D., Hames B. D. Edited by Washington, D.C.: IRL Press;
    [Google Scholar]
  23. Mark D. F., Lu S. D., Creasey A. A., Yamamoto R., Lin L. S. 1984; Site-specific mutagenesis of the human fibroblast interferon gene. Proceedings of the National Academy of Sciences U.S.A: 815662–5666
    [Google Scholar]
  24. Matsumura M., Signor G., Mathews B. W. 1989; Substantial increase of protein stability by multiple disulphide bonds. Nature; London: 342291–293
    [Google Scholar]
  25. Norrby E., Oxman M. 1990; Measles virus. In Virology, 2nd edn.. pp 1013–1044 Fields B. N., Knipe D. M. Edited by New York: Raven Press;
    [Google Scholar]
  26. Norrby E., Chen S. N., Togashi T., Sheshberadaran H., Johnson K. P. 1982; Five measles virus antigens demonstrated by use of mouse hybridoma antibodies in productively infected tissue cells. Archives of Virology 71:1–11
    [Google Scholar]
  27. Nowak T., Wengler G. 1987; Analysis of disulfides present in the membrane proteins of the West Nile flavivirus. Virology 156:127–137
    [Google Scholar]
  28. Pace C. N., Grimsley G. R., Thompson J. A., Barnett B. J. 1988; Conformational stability and activity of ribonucleaseTl with zero, one, and two intact disulphide bond. Journal of Biological Chemistry 263:11820–11825
    [Google Scholar]
  29. Pfeffer S. R., Rothman J. E. 1987; Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annual Review of Biochemistry 56:829–852
    [Google Scholar]
  30. Pinter A., Honne W. J., Tilley S. A., Bona C., Zaghouani H., Gorney M. K., Zolla-Pazner S. 1989; Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1. Journal of Virology 63:2674–2679
    [Google Scholar]
  31. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn.. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  32. Segal M. S., Bye J. M., Sambrook J., Gething M. -J. 1992; Disulphide bond formation during the folding of influenza virus hemagglutinin. Journal of Cell Biology 118:227–244
    [Google Scholar]
  33. Sheshberadaran H., Norrby E. 1986; Characterization of epitopes on the measles virus hemagglutinin. Virology 152:58–65
    [Google Scholar]
  34. Sheshberadaran H., Chen S. H., Norrby E. 1983; Monoclonal antibodies against five structural components of measles virus. I. Characterization of antigenic determinants on nine strains of measles virus. Virology 128:341–353
    [Google Scholar]
  35. Tsukiyama K., Sugiyama M., Yoshikawa Y., Yamanouchi K. 1987; Molecular cloning and sequence analysis of the rinderpest virus mRNA encoding the hemagglutinin protein. Virology 150:48–54
    [Google Scholar]
  36. Wang A., Liu S. D., Mark D. F. 1984; Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues. Science 224:1431–1433
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-75-9-2173
Loading
/content/journal/jgv/10.1099/0022-1317-75-9-2173
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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