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. 1991 Oct;65(10):5323–5332. doi: 10.1128/jvi.65.10.5323-5332.1991

Mutational analysis of N-linked glycosylation sites of Friend murine leukemia virus envelope protein.

S C Kayman 1, R Kopelman 1, S Projan 1, D M Kinney 1, A Pinter 1
PMCID: PMC249012  PMID: 1895386

Abstract

The roles played by the N-linked glycans of the Friend murine leukemia virus envelope proteins were investigated by site-specific mutagenesis. The surface protein gp70 has eight potential attachment sites for N-linked glycan; each signal asparagine was converted to aspartate, and mutant viruses were tested for the ability to grow in NIH 3T3 fibroblasts. Seven of the mutations did not affect virus infectivity, whereas mutation of the fourth glycosylation signal from the amino terminus (gs4) resulted in a noninfectious phenotype. Characterization of mutant gene products by radioimmunoprecipitation confirmed that glycosylation occurs at all eight consensus signals in gp70 and that gs2 carries an endoglycosidase H-sensitive glycan. Elimination of gs2 did not cause retention of an endoglycosidase H-sensitive glycan at a different site, demonstrating that this structure does not play an essential role in envelope protein function. The gs3- mutation affected a second posttranslational modification of unknown type, which was manifested as production of gp70 that remained smaller than wild-type gp70 after removal of all N-linked glycans by peptide N-glycosidase F. The gs4- mutation decreased processing of gPr80 to gPr90, completely inhibited proteolytic processing of gPr90 to gp70 and Pr15(E), and prevented incorporation of envelope products into virus particles. Brefeldin A-induced mixing of the endoplasmic reticulum and parts of the Golgi apparatus allowed proteolytic processing of wild-type gPr90 to occur in the absence of protein transport, but it did not overcome the cleavage defect of the gs4- precursor, indicating that gs4- gPr90 is resistant to the processing protease. The work reported here demonstrates that the gs4 region is important for env precursor processing and suggests that gs4 may be a critical target in the disruption of murine leukemia virus env product processing by inhibitors of N-linked glycosylation.

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Selected References

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

  1. Adachi A., Sakai K., Kitamura N., Nakanishi S., Niwa O., Matsuyama M., Ishimoto A. Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA. J Virol. 1984 Jun;50(3):813–821. doi: 10.1128/jvi.50.3.813-821.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bestwick R. K., Kozak S. L., Kabat D. Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5404–5408. doi: 10.1073/pnas.85.15.5404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bosselman R. A., van Straaten F., Van Beveren C., Verma I. M., Vogt M. Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA. J Virol. 1982 Oct;44(1):19–31. doi: 10.1128/jvi.44.1.19-31.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chege N. W., Pfeffer S. R. Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network. J Cell Biol. 1990 Sep;111(3):893–899. doi: 10.1083/jcb.111.3.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen R. Complete amino acid sequence and glycosylation sites of glycoprotein gp71A of Friend murine leukemia virus. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5788–5792. doi: 10.1073/pnas.79.19.5788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chesebro B., Wehrly K., Cloyd M., Britt W., Portis J., Collins J., Nishio J. Characterization of mouse monoclonal antibodies specific for Friend murine leukemia virus-induced erythroleukemia cells: friend-specific and FMR-specific antigens. Virology. 1981 Jul 15;112(1):131–144. doi: 10.1016/0042-6822(81)90619-x. [DOI] [PubMed] [Google Scholar]
  7. Delassus S., Sonigo P., Wain-Hobson S. Genetic organization of gibbon ape leukemia virus. Virology. 1989 Nov;173(1):205–213. doi: 10.1016/0042-6822(89)90236-5. [DOI] [PubMed] [Google Scholar]
  8. Dorner A. J., Bole D. G., Kaufman R. J. The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J Cell Biol. 1987 Dec;105(6 Pt 1):2665–2674. doi: 10.1083/jcb.105.6.2665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubé S., Fisher J. W., Powell J. S. Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function. J Biol Chem. 1988 Nov 25;263(33):17516–17521. [PubMed] [Google Scholar]
  10. Elder J. H., Mullins J. I. Nucleotide sequence of the envelope gene of Gardner-Arnstein feline leukemia virus B reveals unique sequence homologies with a murine mink cell focus-forming virus. J Virol. 1983 Jun;46(3):871–880. doi: 10.1128/jvi.46.3.871-880.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  12. Geyer R., Dabrowski J., Dabrowski U., Linder D., Schlüter M., Schott H. H., Stirm S. Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus. Eur J Biochem. 1990 Jan 12;187(1):95–110. doi: 10.1111/j.1432-1033.1990.tb15281.x. [DOI] [PubMed] [Google Scholar]
  13. Holland C. A., Wozney J., Hopkins N. Nucleotide sequence of the gp70 gene of murine retrovirus MCF 247. J Virol. 1983 Sep;47(3):413–420. doi: 10.1128/jvi.47.3.413-420.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koch W., Hunsmann G., Friedrich R. Nucleotide sequence of the envelope gene of Friend murine leukemia virus. J Virol. 1983 Jan;45(1):1–9. doi: 10.1128/jvi.45.1.1-9.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Koch W., Zimmermann W., Oliff A., Friedrich R. Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-inducing virus: implications for the functions of these sequences. J Virol. 1984 Mar;49(3):828–840. doi: 10.1128/jvi.49.3.828-840.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lenz J., Crowther R., Straceski A., Haseltine W. Nucleotide sequence of the Akv env gene. J Virol. 1982 May;42(2):519–529. doi: 10.1128/jvi.42.2.519-529.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lostrom M. E., Stone M. R., Tam M., Burnette W. N., Pinter A., Nowinski R. C. Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants on the p 15(E) and gp70 envelope proteins. Virology. 1979 Oct 30;98(2):336–350. doi: 10.1016/0042-6822(79)90557-9. [DOI] [PubMed] [Google Scholar]
  21. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. McClary J. A., Witney F., Geisselsoder J. Efficient site-directed in vitro mutagenesis using phagemid vectors. Biotechniques. 1989 Mar;7(3):282–289. [PubMed] [Google Scholar]
  24. Merregaert J., Nuyten J. M., Janowski M. Nucleotide sequence of the envelope gene of radiation leukemia virus. Virology. 1985 Jul 30;144(2):457–467. doi: 10.1016/0042-6822(85)90286-7. [DOI] [PubMed] [Google Scholar]
  25. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Misumi Y., Misumi Y., Miki K., Takatsuki A., Tamura G., Ikehara Y. Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J Biol Chem. 1986 Aug 25;261(24):11398–11403. [PubMed] [Google Scholar]
  27. Morikawa Y., Moore J. P., Wilkinson A. J., Jones I. M. Reduction in CD4 binding affinity associated with removal of a single glycosylation site in the external glycoprotein of HIV-2. Virology. 1991 Feb;180(2):853–856. doi: 10.1016/0042-6822(91)90106-l. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. O'Donnell P. V., Nowinski R. C. Serological analysis of antigenic determinants on the env gene products of AKR dualtropic (MCF) murine leukemia viruses. Virology. 1980 Nov;107(1):81–88. doi: 10.1016/0042-6822(80)90274-3. [DOI] [PubMed] [Google Scholar]
  30. O'Neill R. R., Buckler C. E., Theodore T. S., Martin M. A., Repaske R. Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus. J Virol. 1985 Jan;53(1):100–106. doi: 10.1128/jvi.53.1.100-106.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Oda T., Ikeda S., Watanabe S., Hatsushika M., Akiyama K., Mitsunobu F. Molecular cloning, complete nucleotide sequence, and gene structure of the provirus genome of a retrovirus produced in a human lymphoblastoid cell line. Virology. 1988 Dec;167(2):468–476. [PubMed] [Google Scholar]
  32. Oliff A. I., Hager G. L., Chang E. H., Scolnick E. M., Chan H. W., Lowy D. R. Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus. J Virol. 1980 Jan;33(1):475–486. doi: 10.1128/jvi.33.1.475-486.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ott D., Friedrich R., Rein A. Sequence analysis of amphotropic and 10A1 murine leukemia viruses: close relationship to mink cell focus-inducing viruses. J Virol. 1990 Feb;64(2):757–766. doi: 10.1128/jvi.64.2.757-766.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Overbaugh J., Donahue P. R., Quackenbush S. L., Hoover E. A., Mullins J. I. Molecular cloning of a feline leukemia virus that induces fatal immunodeficiency disease in cats. Science. 1988 Feb 19;239(4842):906–910. doi: 10.1126/science.2893454. [DOI] [PubMed] [Google Scholar]
  35. Pierotti M., DeLeo A. B., Pinter A., O'Donnell P. V., Hämmerling U., Fleissner E. The GIX antigen of murine leukemia virus: an analysis with monoclonal antibodies. Virology. 1981 Jul 30;112(2):450–460. doi: 10.1016/0042-6822(81)90292-0. [DOI] [PubMed] [Google Scholar]
  36. Pinter A., Honnen W. J. Characterization of structural and immunological properties of specific domains of Friend ecotropic and dual-tropic murine leukemia virus gp70s. J Virol. 1984 Feb;49(2):452–458. doi: 10.1128/jvi.49.2.452-458.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pinter A., Honnen W. J. Comparison of structural domains of gp70s of ecotropic Akv and dualtropic MCF-247 MuLVs. Virology. 1983 Aug;129(1):40–50. doi: 10.1016/0042-6822(83)90394-x. [DOI] [PubMed] [Google Scholar]
  38. Pinter A., Honnen W. J., Li J. S. Studies with inhibitors of oligosaccharide processing indicate a functional role for complex sugars in the transport and proteolysis of Friend mink cell focus-inducing murine leukemia virus envelope proteins. Virology. 1984 Jul 15;136(1):196–210. doi: 10.1016/0042-6822(84)90259-9. [DOI] [PubMed] [Google Scholar]
  39. Pinter A., Honnen W. J. O-linked glycosylation of retroviral envelope gene products. J Virol. 1988 Mar;62(3):1016–1021. doi: 10.1128/jvi.62.3.1016-1021.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pitta A. M., Rose J. K., Machamer C. E. A single-amino-acid substitution eliminates the stringent carbohydrate requirement for intracellular transport of a viral glycoprotein. J Virol. 1989 Sep;63(9):3801–3809. doi: 10.1128/jvi.63.9.3801-3809.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Polonoff E., Machida C. A., Kabat D. Glycosylation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acid analogues and by tunicamycin. J Biol Chem. 1982 Dec 10;257(23):14023–14028. [PubMed] [Google Scholar]
  42. Power M. D., Marx P. A., Bryant M. L., Gardner M. B., Barr P. J., Luciw P. A. Nucleotide sequence of SRV-1, a type D simian acquired immune deficiency syndrome retrovirus. Science. 1986 Mar 28;231(4745):1567–1572. doi: 10.1126/science.3006247. [DOI] [PubMed] [Google Scholar]
  43. Rassart E., Nelbach L., Jolicoeur P. Cas-Br-E murine leukemia virus: sequencing of the paralytogenic region of its genome and derivation of specific probes to study its origin and the structure of its recombinant genomes in leukemic tissues. J Virol. 1986 Dec;60(3):910–919. doi: 10.1128/jvi.60.3.910-919.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Riedel N., Hoover E. A., Gasper P. W., Nicolson M. O., Mullins J. I. Molecular analysis and pathogenesis of the feline aplastic anemia retrovirus, feline leukemia virus C-Sarma. J Virol. 1986 Oct;60(1):242–250. doi: 10.1128/jvi.60.1.242-250.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Schlüter M., Linder D., Geyer R., Hunsmann G., Schneider J., Stirm S. The glycoprotein 71 of ecotropic Friend murine leukemia virus. Structure of the oligosaccharides linked to asparagine-12. FEBS Lett. 1984 Apr 24;169(2):194–198. doi: 10.1016/0014-5793(84)80317-8. [DOI] [PubMed] [Google Scholar]
  47. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  48. Sitbon M., Ellerbrok H., Pozo F., Nishio J., Hayes S. F., Evans L. H., Chesebro B. Sequences in the U5-gag-pol region influence early and late pathogenic effects of Friend and Moloney murine leukemia viruses. J Virol. 1990 May;64(5):2135–2140. doi: 10.1128/jvi.64.5.2135-2140.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Skehel J. J., Stevens D. J., Daniels R. S., Douglas A. R., Knossow M., Wilson I. A., Wiley D. C. A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1779–1783. doi: 10.1073/pnas.81.6.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sodora D. L., Cohen G. H., Eisenberg R. J. Influence of asparagine-linked oligosaccharides on antigenicity, processing, and cell surface expression of herpes simplex virus type 1 glycoprotein D. J Virol. 1989 Dec;63(12):5184–5193. doi: 10.1128/jvi.63.12.5184-5193.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sonigo P., Barker C., Hunter E., Wain-Hobson S. Nucleotide sequence of Mason-Pfizer monkey virus: an immunosuppressive D-type retrovirus. Cell. 1986 May 9;45(3):375–385. doi: 10.1016/0092-8674(86)90323-5. [DOI] [PubMed] [Google Scholar]
  52. Stewart M. A., Warnock M., Wheeler A., Wilkie N., Mullins J. I., Onions D. E., Neil J. C. Nucleotide sequences of a feline leukemia virus subgroup A envelope gene and long terminal repeat and evidence for the recombinational origin of subgroup B viruses. J Virol. 1986 Jun;58(3):825–834. doi: 10.1128/jvi.58.3.825-834.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Szurek P. F., Yuen P. H., Ball J. K., Wong P. K. A Val-25-to-Ile substitution in the envelope precursor polyprotein, gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env, and neurovirulence of ts1, a mutant of Moloney murine leukemia virus TB. J Virol. 1990 Feb;64(2):467–475. doi: 10.1128/jvi.64.2.467-475.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Thayer R. M., Power M. D., Bryant M. L., Gardner M. B., Barr P. J., Luciw P. A. Sequence relationships of type D retroviruses which cause simian acquired immunodeficiency syndrome. Virology. 1987 Apr;157(2):317–329. doi: 10.1016/0042-6822(87)90274-1. [DOI] [PubMed] [Google Scholar]
  57. Trimble R. B., Maley F. Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H. Anal Biochem. 1984 Sep;141(2):515–522. doi: 10.1016/0003-2697(84)90080-0. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Vogt M., Haggblom C., Swift S., Haas M. Envelope gene and long terminal repeat determine the different biological properties of Rauscher, Friend, and Moloney mink cell focus-inducing viruses. J Virol. 1985 Jul;55(1):184–192. doi: 10.1128/jvi.55.1.184-192.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Voytek P., Kozak C. A. Nucleotide sequence and mode of transmission of the wild mouse ecotropic virus, HoMuLV. Virology. 1989 Nov;173(1):58–67. doi: 10.1016/0042-6822(89)90221-3. [DOI] [PubMed] [Google Scholar]
  61. Wilhelmsen K. C., Eggleton K., Temin H. M. Nucleic acid sequences of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homolog, the proto-oncogene c-rel. J Virol. 1984 Oct;52(1):172–182. doi: 10.1128/jvi.52.1.172-182.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Willey R. L., Smith D. H., Lasky L. A., Theodore T. S., Earl P. L., Moss B., Capon D. J., Martin M. A. In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity. J Virol. 1988 Jan;62(1):139–147. doi: 10.1128/jvi.62.1.139-147.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wolff L., Ruscetti S. The spleen focus-forming virus (SFFV) envelope gene, when introduced into mice in the absence of other SFFV genes, induces acute erythroleukemia. J Virol. 1988 Jun;62(6):2158–2163. doi: 10.1128/jvi.62.6.2158-2163.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wright K. E., Salvato M. S., Buchmeier M. J. Neutralizing epitopes of lymphocytic choriomeningitis virus are conformational and require both glycosylation and disulfide bonds for expression. Virology. 1989 Aug;171(2):417–426. doi: 10.1016/0042-6822(89)90610-7. [DOI] [PubMed] [Google Scholar]

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