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. 1989 Dec;63(12):5184–5193. doi: 10.1128/jvi.63.12.5184-5193.1989

Influence of asparagine-linked oligosaccharides on antigenicity, processing, and cell surface expression of herpes simplex virus type 1 glycoprotein D.

D L Sodora 1, G H Cohen 1, R J Eisenberg 1
PMCID: PMC251182  PMID: 2555549

Abstract

Glycoprotein D (gD) is an envelope component of herpes simplex virus types 1 and 2. gD-1 contains three sites for the addition of N-linked carbohydrate (N-CHO), all of which are used. Three mutants were constructed by site-directed mutagenesis, each of which altered one N-CHO addition site from Asn-X-Thr/Ser to Asn-X-Ala. A fourth mutant was altered at all three sites. The mutant genes were inserted into an expression vector, and the expressed protein was analyzed in transiently transfected COS-1 cells. The mutant protein lacking N-CHO at site 1 (Asn-94) had a reduced affinity for monoclonal antibodies (MAbs) to discontinuous epitopes, suggesting that the conformation of the protein had been altered. However, the protein was processed and transported to the cell surface. The absence of N-CHO at site 2 (Asn-121) had no apparent effect on processing or transport of gD-1 but resulted in reduced binding of two MAbs previously shown to be in group VI. Binding of other MAbs to discontinuous epitopes (including other group VI MAbs) was not affected. The absence of N-CHO at site 3 (Asn-262) had no effect on processing, transport, or conformation of the gD-1 protein. The absence of N-CHO from site 1 or from all three sites resulted in the formation of high-molecular-weight aggregates or complexes and a reduction in MAb binding. However, these proteins were modified by the addition of O-glycans and transported to the cell surface. We conclude that the absence of the first or all N-linked carbohydrates alters the native conformation of gD-1 but does not prevent its transport to the cell surface.

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Selected References

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

  1. Barbosa J. A., Santos-Aguado J., Mentzer S. J., Strominger J. L., Burakoff S. J., Biro P. A. Site-directed mutagenesis of class I HLA genes. Role of glycosylation in surface expression and functional recognition. J Exp Med. 1987 Nov 1;166(5):1329–1350. doi: 10.1084/jem.166.5.1329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  3. Cohen G. H., Dietzschold B., Ponce de Leon M., Long D., Golub E., Varrichio A., Pereira L., Eisenberg R. J. Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody. J Virol. 1984 Jan;49(1):102–108. doi: 10.1128/jvi.49.1.102-108.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cohen G. H., Isola V. J., Kuhns J., Berman P. W., Eisenberg R. J. Localization of discontinuous epitopes of herpes simplex virus glycoprotein D: use of a nondenaturing ("native" gel) system of polyacrylamide gel electrophoresis coupled with Western blotting. J Virol. 1986 Oct;60(1):157–166. doi: 10.1128/jvi.60.1.157-166.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen G. H., Katze M., Hydrean-Stern C., Eisenberg R. J. Type-common CP-1 antigen of herpes simplex virus is associated with a 59,000-molecular-weight envelope glycoprotein. J Virol. 1978 Jul;27(1):172–181. doi: 10.1128/jvi.27.1.172-181.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen G. H., Long D., Eisenberg R. J. Synthesis and processing of glycoproteins gD and gC of herpes simplex virus type 1. J Virol. 1980 Nov;36(2):429–439. doi: 10.1128/jvi.36.2.429-439.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen G. H., Long D., Matthews J. T., May M., Eisenberg R. Glycopeptides of the type-common glycoprotein gD of herpes simplex virus types 1 and 2. J Virol. 1983 Jun;46(3):679–689. doi: 10.1128/jvi.46.3.679-689.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen G. H., Wilcox W. C., Sodora D. L., Long D., Levin J. Z., Eisenberg R. J. Expression of herpes simplex virus type 1 glycoprotein D deletion mutants in mammalian cells. J Virol. 1988 Jun;62(6):1932–1940. doi: 10.1128/jvi.62.6.1932-1940.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crawford D. H., Callard R. E., Muggeridge M. I., Mitchell D. M., Zanders E. D., Beverley P. C. Production of human monoclonal antibody to X31 influenza virus nucleoprotein. J Gen Virol. 1983 Mar;64(Pt 3):697–700. doi: 10.1099/0022-1317-64-3-697. [DOI] [PubMed] [Google Scholar]
  10. Doms R. W., Ruusala A., Machamer C., Helenius J., Helenius A., Rose J. K. Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein. J Cell Biol. 1988 Jul;107(1):89–99. doi: 10.1083/jcb.107.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eisenberg R. J., Hydrean-Stern C., Cohen G. H. Structural analysis of precursor and product forms of type-common envelope glycoprotein D (CP-1 antigen) of herpes simplex virus type 1. J Virol. 1979 Sep;31(3):608–620. doi: 10.1128/jvi.31.3.608-620.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eisenberg R. J., Long D., Hogue-Angeletti R., Cohen G. H. Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2. J Virol. 1984 Jan;49(1):265–268. doi: 10.1128/jvi.49.1.265-268.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eisenberg R. J., Long D., Pereira L., Hampar B., Zweig M., Cohen G. H. Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1. J Virol. 1982 Feb;41(2):478–488. doi: 10.1128/jvi.41.2.478-488.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eisenberg R. J., Long D., Ponce de Leon M., Matthews J. T., Spear P. G., Gibson M. G., Lasky L. A., Berman P., Golub E., Cohen G. H. Localization of epitopes of herpes simplex virus type 1 glycoprotein D. J Virol. 1985 Feb;53(2):634–644. doi: 10.1128/jvi.53.2.634-644.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Eisenberg R. J., Ponce de Leon M., Pereira L., Long D., Cohen G. H. Purification of glycoprotein gD of herpes simplex virus types 1 and 2 by use of monoclonal antibody. J Virol. 1982 Mar;41(3):1099–1104. doi: 10.1128/jvi.41.3.1099-1104.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Elder J. H., Alexander S. endo-beta-N-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4540–4544. doi: 10.1073/pnas.79.15.4540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fuller A. O., Spear P. G. Anti-glycoprotein D antibodies that permit adsorption but block infection by herpes simplex virus 1 prevent virion-cell fusion at the cell surface. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5454–5458. doi: 10.1073/pnas.84.15.5454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fuller A. O., Spear P. G. Specificities of monoclonal and polyclonal antibodies that inhibit adsorption of herpes simplex virus to cells and lack of inhibition by potent neutralizing antibodies. J Virol. 1985 Aug;55(2):475–482. doi: 10.1128/jvi.55.2.475-482.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gibson R., Schlesinger S., Kornfeld S. The nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures. J Biol Chem. 1979 May 10;254(9):3600–3607. [PubMed] [Google Scholar]
  20. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Highlander S. L., Sutherland S. L., Gage P. J., Johnson D. C., Levine M., Glorioso J. C. Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration. J Virol. 1987 Nov;61(11):3356–3364. doi: 10.1128/jvi.61.11.3356-3364.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Isola V. J., Eisenberg R. J., Siebert G. R., Heilman C. J., Wilcox W. C., Cohen G. H. Fine mapping of antigenic site II of herpes simplex virus glycoprotein D. J Virol. 1989 May;63(5):2325–2334. doi: 10.1128/jvi.63.5.2325-2334.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Johnson D. C., Ligas M. W. Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors. J Virol. 1988 Dec;62(12):4605–4612. doi: 10.1128/jvi.62.12.4605-4612.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Johnson D. C., Smiley J. R. Intracellular transport of herpes simplex virus gD occurs more rapidly in uninfected cells than in infected cells. J Virol. 1985 Jun;54(3):682–689. doi: 10.1128/jvi.54.3.682-689.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Johnson D. C., Spear P. G. O-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the Golgi apparatus. Cell. 1983 Mar;32(3):987–997. doi: 10.1016/0092-8674(83)90083-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kozarsky K. F., Call S. M., Dower S. K., Krieger M. Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors. Mol Cell Biol. 1988 Aug;8(8):3357–3363. doi: 10.1128/mcb.8.8.3357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Kühn J. E., Eing B. R., Brossmer R., Munk K., Braun R. W. Removal of N-linked carbohydrates decreases the infectivity of herpes simplex virus type 1. J Gen Virol. 1988 Nov;69(Pt 11):2847–2858. doi: 10.1099/0022-1317-69-11-2847. [DOI] [PubMed] [Google Scholar]
  29. Lasky L. A., Dowbenko D. J. DNA sequence analysis of the type-common glycoprotein-D genes of herpes simplex virus types 1 and 2. DNA. 1984;3(1):23–29. doi: 10.1089/dna.1.1984.3.23. [DOI] [PubMed] [Google Scholar]
  30. Leavitt R., Schlesinger S., Kornfeld S. Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus. J Biol Chem. 1977 Dec 25;252(24):9018–9023. [PubMed] [Google Scholar]
  31. Ligas M. W., Johnson D. C. A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells. J Virol. 1988 May;62(5):1486–1494. doi: 10.1128/jvi.62.5.1486-1494.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Long D., Madara T. J., Ponce de Leon M., Cohen G. H., Montgomery P. C., Eisenberg R. J. Glycoprotein D protects mice against lethal challenge with herpes simplex virus types 1 and 2. Infect Immun. 1984 Feb;43(2):761–764. doi: 10.1128/iai.43.2.761-764.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Matthews J. T., Cohen G. H., Eisenberg R. J. Synthesis and processing of glycoprotein D of herpes simplex virus types 1 and 2 in an in vitro system. J Virol. 1983 Nov;48(2):521–533. doi: 10.1128/jvi.48.2.521-533.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Matzuk M. M., Boime I. Site-specific mutagenesis defines the intracellular role of the asparagine-linked oligosaccharides of chorionic gonadotropin beta subunit. J Biol Chem. 1988 Nov 15;263(32):17106–17111. [PubMed] [Google Scholar]
  38. 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]
  39. Minson A. C., Hodgman T. C., Digard P., Hancock D. C., Bell S. E., Buckmaster E. A. An analysis of the biological properties of monoclonal antibodies against glycoprotein D of herpes simplex virus and identification of amino acid substitutions that confer resistance to neutralization. J Gen Virol. 1986 Jun;67(Pt 6):1001–1013. doi: 10.1099/0022-1317-67-6-1001. [DOI] [PubMed] [Google Scholar]
  40. Muggeridge M. I., Isola V. J., Byrn R. A., Tucker T. J., Minson A. C., Glorioso J. C., Cohen G. H., Eisenberg R. J. Antigenic analysis of a major neutralization site of herpes simplex virus glycoprotein D, using deletion mutants and monoclonal antibody-resistant mutants. J Virol. 1988 Sep;62(9):3274–3280. doi: 10.1128/jvi.62.9.3274-3280.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Noble A. G., Lee G. T., Sprague R., Parish M. L., Spear P. G. Anti-gD monoclonal antibodies inhibit cell fusion induced by herpes simplex virus type 1. Virology. 1983 Aug;129(1):218–224. doi: 10.1016/0042-6822(83)90409-9. [DOI] [PubMed] [Google Scholar]
  42. Norrild B., Pedersen B. Effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and their expression on the cell surface. J Virol. 1982 Aug;43(2):395–402. doi: 10.1128/jvi.43.2.395-402.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Peake M. L., Nystrom P., Pizer L. I. Herpesvirus glycoprotein synthesis and insertion into plasma membranes. J Virol. 1982 May;42(2):678–690. doi: 10.1128/jvi.42.2.678-690.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Petrovskis E. A., Timmins J. G., Armentrout M. A., Marchioli C. C., Yancey R. J., Jr, Post L. E. DNA sequence of the gene for pseudorabies virus gp50, a glycoprotein without N-linked glycosylation. J Virol. 1986 Aug;59(2):216–223. doi: 10.1128/jvi.59.2.216-223.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Pizer L. I., Cohen G. H., Eisenberg R. J. Effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production. J Virol. 1980 Apr;34(1):142–153. doi: 10.1128/jvi.34.1.142-153.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rothman J. E. Protein sorting by selective retention in the endoplasmic reticulum and Golgi stack. Cell. 1987 Aug 14;50(4):521–522. doi: 10.1016/0092-8674(87)90024-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. Serafini-Cessi F., Dall'Olio F., Malagolini N., Pereira L., Campadelli-Fiume G. Comparative study on O-linked oligosaccharides of glycoprotein D of herpes simplex virus types 1 and 2. J Gen Virol. 1988 Apr;69(Pt 4):869–877. doi: 10.1099/0022-1317-69-4-869. [DOI] [PubMed] [Google Scholar]
  51. Showalter S. D., Zweig M., Hampar B. Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP 4. Infect Immun. 1981 Dec;34(3):684–692. doi: 10.1128/iai.34.3.684-692.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Spear P. G. Membrane proteins specified by herpes simplex viruses. I. Identification of four glycoprotein precursors and their products in type 1-infected cells. J Virol. 1976 Mar;17(3):991–1008. doi: 10.1128/jvi.17.3.991-1008.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Spivack J. G., Prusoff W. H., Tritton T. R. A study of the antiviral mechanism of action of 2-deoxy-D-glucose: normally glycosylated proteins are not strictly required for herpes simplex virus attachment but increase viral penetration and infectivity. Virology. 1982 Nov;123(1):123–138. doi: 10.1016/0042-6822(82)90300-2. [DOI] [PubMed] [Google Scholar]
  54. Svennerholm B., Olofsson S., Lundén R., Vahlne A., Lycke E. Adsorption and penetration of enveloped herpes simplex virus particles modified by tunicamycin or 2-deoxy-D-glucose. J Gen Virol. 1982 Dec;63(2):343–349. doi: 10.1099/0022-1317-63-2-343. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. 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]
  57. Watson R. J. DNA sequence of the Herpes simplex virus type 2 glycoprotein D gene. Gene. 1983 Dec;26(2-3):307–312. doi: 10.1016/0378-1119(83)90203-2. [DOI] [PubMed] [Google Scholar]
  58. Watson R. J., Weis J. H., Salstrom J. S., Enquist L. W. Herpes simplex virus type-1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli. Science. 1982 Oct 22;218(4570):381–384. doi: 10.1126/science.6289440. [DOI] [PubMed] [Google Scholar]
  59. Wilcox W. C., Long D., Sodora D. L., Eisenberg R. J., Cohen G. H. The contribution of cysteine residues to antigenicity and extent of processing of herpes simplex virus type 1 glycoprotein D. J Virol. 1988 Jun;62(6):1941–1947. doi: 10.1128/jvi.62.6.1941-1947.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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