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. 1994 Mar;68(3):1522–1531. doi: 10.1128/jvi.68.3.1522-1531.1994

Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

G Alkhatib 1, S H Shen 1, D Briedis 1, C Richardson 1, B Massie 1, R Weinberg 1, D Smith 1, J Taylor 1, E Paoletti 1, J Roder 1
PMCID: PMC236609  PMID: 8107215

Abstract

The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alkhatib G., Briedis D. J. High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system. J Virol. 1988 Aug;62(8):2718–2727. doi: 10.1128/jvi.62.8.2718-2727.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alkhatib G., Briedis D. J. The predicted primary structure of the measles virus hemagglutinin. Virology. 1986 Apr 30;150(2):479–490. doi: 10.1016/0042-6822(86)90312-0. [DOI] [PubMed] [Google Scholar]
  3. Alkhatib G., Richardson C., Shen S. H. Intracellular processing, glycosylation, and cell-surface expression of the measles virus fusion protein (F) encoded by a recombinant adenovirus. Virology. 1990 Mar;175(1):262–270. doi: 10.1016/0042-6822(90)90207-8. [DOI] [PubMed] [Google Scholar]
  4. Blumberg B. M., Giorgi C., Rose K., Kolakofsky D. Sequence determination of the Sendai virus fusion protein gene. J Gen Virol. 1985 Feb;66(Pt 2):317–331. doi: 10.1099/0022-1317-66-2-317. [DOI] [PubMed] [Google Scholar]
  5. Bosch M. L., Earl P. L., Fargnoli K., Picciafuoco S., Giombini F., Wong-Staal F., Franchini G. Identification of the fusion peptide of primate immunodeficiency viruses. Science. 1989 May 12;244(4905):694–697. doi: 10.1126/science.2541505. [DOI] [PubMed] [Google Scholar]
  6. Choppin P. W., Scheid A. The role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses. Rev Infect Dis. 1980 Jan-Feb;2(1):40–61. doi: 10.1093/clinids/2.1.40. [DOI] [PubMed] [Google Scholar]
  7. Deshpande K. L., Fried V. A., Ando M., Webster R. G. Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc Natl Acad Sci U S A. 1987 Jan;84(1):36–40. doi: 10.1073/pnas.84.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dewar R. L., Vasudevachari M. B., Natarajan V., Salzman N. P. Biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport. J Virol. 1989 Jun;63(6):2452–2456. doi: 10.1128/jvi.63.6.2452-2456.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Doms R. W., Lamb R. A., Rose J. K., Helenius A. Folding and assembly of viral membrane proteins. Virology. 1993 Apr;193(2):545–562. doi: 10.1006/viro.1993.1164. [DOI] [PubMed] [Google Scholar]
  10. Elango N., Varsanyi T. M., Kövamees J., Norrby E. The mumps virus fusion protein mRNA sequence and homology among the paramyxoviridae proteins. J Gen Virol. 1989 Apr;70(Pt 4):801–807. doi: 10.1099/0022-1317-70-4-801. [DOI] [PubMed] [Google Scholar]
  11. Gallagher P. J., Henneberry J. M., Sambrook J. F., Gething M. J. Glycosylation requirements for intracellular transport and function of the hemagglutinin of influenza virus. J Virol. 1992 Dec;66(12):7136–7145. doi: 10.1128/jvi.66.12.7136-7145.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Graves M. C., Silver S. M., Choppin P. W. Measles virus polypeptides synthesis in infected cells. Virology. 1978 May 1;86(1):254–263. doi: 10.1016/0042-6822(78)90025-9. [DOI] [PubMed] [Google Scholar]
  13. Groenink M., Fouchier R. A., Broersen S., Baker C. H., Koot M., van't Wout A. B., Huisman H. G., Miedema F., Tersmette M., Schuitemaker H. Relation of phenotype evolution of HIV-1 to envelope V2 configuration. Science. 1993 Jun 4;260(5113):1513–1516. doi: 10.1126/science.8502996. [DOI] [PubMed] [Google Scholar]
  14. Guan J. L., Machamer C. E., Rose J. K. Glycosylation allows cell-surface transport of an anchored secretory protein. Cell. 1985 Sep;42(2):489–496. doi: 10.1016/0092-8674(85)90106-0. [DOI] [PubMed] [Google Scholar]
  15. Horvath C. M., Paterson R. G., Shaughnessy M. A., Wood R., Lamb R. A. Biological activity of paramyxovirus fusion proteins: factors influencing formation of syncytia. J Virol. 1992 Jul;66(7):4564–4569. doi: 10.1128/jvi.66.7.4564-4569.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jacoby D. R., Cooke C., Prabakaran I., Boland J., Nathanson N., Gonzalez-Scarano F. Expression of the La Crosse M segment proteins in a recombinant vaccinia expression system mediates pH-dependent cellular fusion. Virology. 1993 Apr;193(2):993–996. doi: 10.1006/viro.1993.1213. [DOI] [PubMed] [Google Scholar]
  17. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lamb R. A. Paramyxovirus fusion: a hypothesis for changes. Virology. 1993 Nov;197(1):1–11. doi: 10.1006/viro.1993.1561. [DOI] [PubMed] [Google Scholar]
  20. Leavitt R., Schlesinger S., Kornfeld S. Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses. J Virol. 1977 Jan;21(1):375–385. doi: 10.1128/jvi.21.1.375-385.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Machamer C. E., Florkiewicz R. Z., Rose J. K. A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface. Mol Cell Biol. 1985 Nov;5(11):3074–3083. doi: 10.1128/mcb.5.11.3074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  23. Morrison T., Ward L. J., Semerjian A. Intracellular processing of the Newcastle disease virus fusion glycoprotein. J Virol. 1985 Mar;53(3):851–857. doi: 10.1128/jvi.53.3.851-857.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ng D. T., Hiebert S. W., Lamb R. A. Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase. Mol Cell Biol. 1990 May;10(5):1989–2001. doi: 10.1128/mcb.10.5.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Olden K., Parent J. B., White S. L. Carbohydrate moieties of glycoproteins. A re-evaluation of their function. Biochim Biophys Acta. 1982 May 12;650(4):209–232. doi: 10.1016/0304-4157(82)90017-x. [DOI] [PubMed] [Google Scholar]
  26. Overton W. R. Modified histogram subtraction technique for analysis of flow cytometry data. Cytometry. 1988 Nov;9(6):619–626. doi: 10.1002/cyto.990090617. [DOI] [PubMed] [Google Scholar]
  27. Paterson R. G., Harris T. J., Lamb R. A. Fusion protein of the paramyxovirus simian virus 5: nucleotide sequence of mRNA predicts a highly hydrophobic glycoprotein. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6706–6710. doi: 10.1073/pnas.81.21.6706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Paterson R. G., Lamb R. A. Ability of the hydrophobic fusion-related external domain of a paramyxovirus F protein to act as a membrane anchor. Cell. 1987 Feb 13;48(3):441–452. doi: 10.1016/0092-8674(87)90195-4. [DOI] [PubMed] [Google Scholar]
  29. Perkus M. E., Limbach K., Paoletti E. Cloning and expression of foreign genes in vaccinia virus, using a host range selection system. J Virol. 1989 Sep;63(9):3829–3836. doi: 10.1128/jvi.63.9.3829-3836.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Richardson C. D., Berkovich A., Rozenblatt S., Bellini W. J. Use of antibodies directed against synthetic peptides for identifying cDNA clones, establishing reading frames, and deducing the gene order of measles virus. J Virol. 1985 Apr;54(1):186–193. doi: 10.1128/jvi.54.1.186-193.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Richardson C., Hull D., Greer P., Hasel K., Berkovich A., Englund G., Bellini W., Rima B., Lazzarini R. The nucleotide sequence of the mRNA encoding the fusion protein of measles virus (Edmonston strain): a comparison of fusion proteins from several different paramyxoviruses. Virology. 1986 Dec;155(2):508–523. doi: 10.1016/0042-6822(86)90212-6. [DOI] [PubMed] [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Santos-Aguado J., Biro P. A., Fuhrmann U., Strominger J. L., Barbosa J. A. Amino acid sequences in the alpha 1 domain and not glycosylation are important in HLA-A2/beta 2-microglobulin association and cell surface expression. Mol Cell Biol. 1987 Mar;7(3):982–990. doi: 10.1128/mcb.7.3.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Scheid A., Choppin P. W. Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity of proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology. 1974 Feb;57(2):475–490. doi: 10.1016/0042-6822(74)90187-1. [DOI] [PubMed] [Google Scholar]
  35. Scheid A., Choppin P. W. Two disulfide-linked polypeptide chains constitute the active F protein of paramyxoviruses. Virology. 1977 Jul 1;80(1):54–66. doi: 10.1016/0042-6822(77)90380-4. [DOI] [PubMed] [Google Scholar]
  36. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tartaglia J., Perkus M. E., Taylor J., Norton E. K., Audonnet J. C., Cox W. I., Davis S. W., van der Hoeven J., Meignier B., Riviere M. NYVAC: a highly attenuated strain of vaccinia virus. Virology. 1992 May;188(1):217–232. doi: 10.1016/0042-6822(92)90752-b. [DOI] [PubMed] [Google Scholar]
  38. Taylor A. K., Wall R. Selective removal of alpha heavy-chain glycosylation sites causes immunoglobulin A degradation and reduced secretion. Mol Cell Biol. 1988 Oct;8(10):4197–4203. doi: 10.1128/mcb.8.10.4197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Taylor J., Pincus S., Tartaglia J., Richardson C., Alkhatib G., Briedis D., Appel M., Norton E., Paoletti E. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge. J Virol. 1991 Aug;65(8):4263–4274. doi: 10.1128/jvi.65.8.4263-4274.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Taylor J., Weinberg R., Tartaglia J., Richardson C., Alkhatib G., Briedis D., Appel M., Norton E., Paoletti E. Nonreplicating viral vectors as potential vaccines: recombinant canarypox virus expressing measles virus fusion (F) and hemagglutinin (HA) glycoproteins. Virology. 1992 Mar;187(1):321–328. doi: 10.1016/0042-6822(92)90321-f. [DOI] [PubMed] [Google Scholar]
  41. Varsanyi T. M., Utter G., Norrby E. Purification, morphology and antigenic characterization of measles virus envelope components. J Gen Virol. 1984 Feb;65(Pt 2):355–366. doi: 10.1099/0022-1317-65-2-355. [DOI] [PubMed] [Google Scholar]
  42. Vialard J., Lalumière M., Vernet T., Briedis D., Alkhatib G., Henning D., Levin D., Richardson C. Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene. J Virol. 1990 Jan;64(1):37–50. doi: 10.1128/jvi.64.1.37-50.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wild T. F., Malvoisin E., Buckland R. Measles virus: both the haemagglutinin and fusion glycoproteins are required for fusion. J Gen Virol. 1991 Feb;72(Pt 2):439–442. doi: 10.1099/0022-1317-72-2-439. [DOI] [PubMed] [Google Scholar]

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