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. 1992 Jun;66(6):3514–3521. doi: 10.1128/jvi.66.6.3514-3521.1992

Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

Z Qiu 1, T C Hobman 1, H L McDonald 1, N O Seto 1, S Gillam 1
PMCID: PMC241132  PMID: 1583721

Abstract

The role of N-linked glycosylation in processing and intracellular transport of rubella virus glycoprotein E2 has been studied by expressing glycosylation mutants of E2 in COS cells. A panel of E2 glycosylation mutants were generated by oligonucleotide-directed mutagenesis. Each of the three potential N-linked glycosylation sites was eliminated separately as well as in combination with the other two sites. Expression of the E2 mutant proteins in COS cells indicated that in rubella virus M33 strain, all three sites are used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability, and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number of deleted carbohydrate sites. The mutant proteins were transported to the endoplasmic reticulum and Golgi complex but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site.

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Selected References

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

  1. Adams G. A., Rose J. K. Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport. Cell. 1985 Jul;41(3):1007–1015. doi: 10.1016/s0092-8674(85)80081-7. [DOI] [PubMed] [Google Scholar]
  2. Andersson S., Davis D. L., Dahlbäck H., Jörnvall H., Russell D. W. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15;264(14):8222–8229. [PubMed] [Google Scholar]
  3. Bardeletti G., Tektoff J., Gautheron D. Rubella virus maturation and production in two host cell systems. Intervirology. 1979;11(2):97–103. doi: 10.1159/000149019. [DOI] [PubMed] [Google Scholar]
  4. Beverley S. M., Wilson A. C. Ancient origin for Hawaiian Drosophilinae inferred from protein comparisons. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4753–4757. doi: 10.1073/pnas.82.14.4753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clarke D. M., Loo T. W., Hui I., Chong P., Gillam S. Nucleotide sequence and in vitro expression of rubella virus 24S subgenomic messenger RNA encoding the structural proteins E1, E2 and C. Nucleic Acids Res. 1987 Apr 10;15(7):3041–3057. doi: 10.1093/nar/15.7.3041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clarke D. M., Loo T. W., McDonald H., Gillam S. Expression of rubella virus cDNA coding for the structural proteins. Gene. 1988 May 15;65(1):23–30. doi: 10.1016/0378-1119(88)90413-1. [DOI] [PubMed] [Google Scholar]
  7. Classification and nomenclature of viruses. Fourth report of the International Committee on Taxonomy of Viruses. Intervirology. 1982;17(1-3):1–199. doi: 10.1159/000149278. [DOI] [PubMed] [Google Scholar]
  8. Dorsett P. H., Miller D. C., Green K. Y., Byrd F. I. Structure and function of the rubella virus proteins. Rev Infect Dis. 1985 Mar-Apr;7 (Suppl 1):S150–S156. doi: 10.1093/clinids/7.supplement_1.s150. [DOI] [PubMed] [Google Scholar]
  9. Earl P. L., Moss B., Doms R. W. Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein. J Virol. 1991 Apr;65(4):2047–2055. doi: 10.1128/jvi.65.4.2047-2055.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elbein A. D. Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu Rev Biochem. 1987;56:497–534. doi: 10.1146/annurev.bi.56.070187.002433. [DOI] [PubMed] [Google Scholar]
  11. Frey T. K., Marr L. D. Sequence of the region coding for virion proteins C and E2 and the carboxy terminus of the nonstructural proteins of rubella virus: comparison with alphaviruses. Gene. 1988;62(1):85–99. doi: 10.1016/0378-1119(88)90582-3. [DOI] [PubMed] [Google Scholar]
  12. Gallagher P., Henneberry J., Wilson I., Sambrook J., Gething M. J. Addition of carbohydrate side chains at novel sites on influenza virus hemagglutinin can modulate the folding, transport, and activity of the molecule. J Cell Biol. 1988 Dec;107(6 Pt 1):2059–2073. doi: 10.1083/jcb.107.6.2059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Green K. Y., Dorsett P. H. Rubella virus antigens: localization of epitopes involved in hemagglutination and neutralization by using monoclonal antibodies. J Virol. 1986 Mar;57(3):893–898. doi: 10.1128/jvi.57.3.893-898.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guan J. L., Cao H., Rose J. K. Cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin alpha subunit. J Biol Chem. 1988 Apr 15;263(11):5306–5313. [PubMed] [Google Scholar]
  15. Hobman T. C., Gillam S. In vitro and in vivo expression of rubella virus glycoprotein E2: the signal peptide is contained in the C-terminal region of capsid protein. Virology. 1989 Nov;173(1):241–250. doi: 10.1016/0042-6822(89)90240-7. [DOI] [PubMed] [Google Scholar]
  16. Hobman T. C., Lundstrom M. L., Gillam S. Processing and intracellular transport of rubella virus structural proteins in COS cells. Virology. 1990 Sep;178(1):122–133. doi: 10.1016/0042-6822(90)90385-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hobman T. C., Qiu Z. Y., Chaye H., Gillam S. Analysis of rubella virus E1 glycosylation mutants expressed in COS cells. Virology. 1991 Apr;181(2):768–772. doi: 10.1016/0042-6822(91)90915-x. [DOI] [PubMed] [Google Scholar]
  18. Hobman T. C., Shukin R., Gillam S. Translocation of rubella virus glycoprotein E1 into the endoplasmic reticulum. J Virol. 1988 Nov;62(11):4259–4264. doi: 10.1128/jvi.62.11.4259-4264.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kundu A., Jabbar M. A., Nayak D. P. Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains. Mol Cell Biol. 1991 May;11(5):2675–2685. doi: 10.1128/mcb.11.5.2675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lundström M. L., Mauracher C. A., Tingle A. J. Characterization of carbohydrates linked to rubella virus glycoprotein E2. J Gen Virol. 1991 Apr;72(Pt 4):843–850. doi: 10.1099/0022-1317-72-4-843. [DOI] [PubMed] [Google Scholar]
  21. Machamer C. E., Rose J. K. Influence of new glycosylation sites on expression of the vesicular stomatitis virus G protein at the plasma membrane. J Biol Chem. 1988 Apr 25;263(12):5948–5954. [PubMed] [Google Scholar]
  22. Machamer C. E., Rose J. K. Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding. J Biol Chem. 1988 Apr 25;263(12):5955–5960. [PubMed] [Google Scholar]
  23. Matzuk M. M., Boime I. The role of the asparagine-linked oligosaccharides of the alpha subunit in the secretion and assembly of human chorionic gonadotrophin. J Cell Biol. 1988 Apr;106(4):1049–1059. doi: 10.1083/jcb.106.4.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Nakhasi H. L., Thomas D., Zheng D. X., Liu T. Y. Nucleotide sequence of capsid, E2 and E1 protein genes of Rubella virus vaccine strain RA27/3. Nucleic Acids Res. 1989 Jun 12;17(11):4393–4394. doi: 10.1093/nar/17.11.4393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Ng D. T., Randall R. E., Lamb R. A. Intracellular maturation and transport of the SV5 type II glycoprotein hemagglutinin-neuraminidase: specific and transient association with GRP78-BiP in the endoplasmic reticulum and extensive internalization from the cell surface. J Cell Biol. 1989 Dec;109(6 Pt 2):3273–3289. doi: 10.1083/jcb.109.6.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Oker-Blom C., Kalkkinen N., Käriäinen L., Pettersson R. F. Rubella virus contains one capsid protein and three envelope glycoproteins, E1, E2a, and E2b. J Virol. 1983 Jun;46(3):964–973. doi: 10.1128/jvi.46.3.964-973.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oker-Blom C. The gene order for rubella virus structural proteins is NH2-C-E2-E1-COOH. J Virol. 1984 Aug;51(2):354–358. doi: 10.1128/jvi.51.2.354-358.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Oker-Blom C., Ulmanen I., Käriäinen L., Pettersson R. F. Rubella virus 40S genome RNA specifies a 24S subgenomic mRNA that codes for a precursor to structural proteins. J Virol. 1984 Feb;49(2):403–408. doi: 10.1128/jvi.49.2.403-408.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  32. Plummer T. H., Jr, Elder J. H., Alexander S., Phelan A. W., Tarentino A. L. Demonstration of peptide:N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F preparations. J Biol Chem. 1984 Sep 10;259(17):10700–10704. [PubMed] [Google Scholar]
  33. Rose J. K., Bergmann J. E. Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell. 1983 Sep;34(2):513–524. doi: 10.1016/0092-8674(83)90384-7. [DOI] [PubMed] [Google Scholar]
  34. Rose J. K., Doms R. W. Regulation of protein export from the endoplasmic reticulum. Annu Rev Cell Biol. 1988;4:257–288. doi: 10.1146/annurev.cb.04.110188.001353. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Tarentino A. L., Maley F. Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J Biol Chem. 1974 Feb 10;249(3):811–817. [PubMed] [Google Scholar]
  37. Tartakoff A. M., Vassalli P. Lectin-binding sites as markers of Golgi subcompartments: proximal-to-distal maturation of oligosaccharides. J Cell Biol. 1983 Oct;97(4):1243–1248. doi: 10.1083/jcb.97.4.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vidal S., Mottet G., Kolakofsky D., Roux L. Addition of high-mannose sugars must precede disulfide bond formation for proper folding of Sendai virus glycoproteins. J Virol. 1989 Feb;63(2):892–900. doi: 10.1128/jvi.63.2.892-900.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vidgren G., Takkinen K., Kalkkinen N., Käriäinen L., Pettersson R. F. Nucleotide sequence of the genes coding for the membrane glycoproteins E1 and E2 of rubella virus. J Gen Virol. 1987 Sep;68(Pt 9):2347–2357. doi: 10.1099/0022-1317-68-9-2347. [DOI] [PubMed] [Google Scholar]
  41. Virtanen I., Ekblom P., Laurila P. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells. J Cell Biol. 1980 May;85(2):429–434. doi: 10.1083/jcb.85.2.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zheng D. X., Dickens L., Liu T. Y., Nakhasi H. L. Nucleotide sequence of the 24S subgenomic messenger RNA of a vaccine strain (HPV77) of rubella virus: comparison with a wild-type strain (M33). Gene. 1989 Oct 30;82(2):343–349. doi: 10.1016/0378-1119(89)90061-9. [DOI] [PubMed] [Google Scholar]
  43. von Bonsdorff C. H., Vaheri A. Growth of rubella virus in BHK21 cells: electron microscopy of morphogenesis. J Gen Virol. 1969 Jul;5(1):47–51. doi: 10.1099/0022-1317-5-1-47. [DOI] [PubMed] [Google Scholar]

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