Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Oct;70(10):6884–6891. doi: 10.1128/jvi.70.10.6884-6891.1996

Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter.

H Wang 1, E Klamo 1, S E Kuhmann 1, S L Kozak 1, M P Kavanaugh 1, D Kabat 1
PMCID: PMC190737  PMID: 8794331

Abstract

The cell surface receptor for ecotropic host-range (infection limited to mice or rats) murine leukemia viruses (MuLVs) is the widely expressed system y+ transporter for cationic amino acids (CAT-1). Like other retroviruses, ecotropic MuLV infection eliminates virus-binding sites from cell surfaces and results in complete interference to superinfection. Surprisingly, infection causes only partial (ca 40 to 60%) loss of mouse CAT-1 transporter activity. The NIH/Swiss mouse CAT-1 (mCAT-1) contains 622 amino acids with 14 hydrophobic potential membrane-spanning sequences, and it is known that the third extracellular loop from the amino terminus is required for virus binding. Although loop 3 is hypervariable in different species and mouse strains, consistent with its proposed role in virus-host coevolution, loop 3 sequences of both susceptible and resistant species contain consensus sites for N-linked glycosylation. Both of the consensus sites in loop 3 of mCAT-1 are known to be glycosylated and to contain oligosaccharides with diverse sizes (J. W. Kim and J. M. Cunningham, J. Biol. Chem. 268:16316-16320, 1993). We confirmed by several lines of evidence that N-linked glycosylation occludes a potentially functional virus-binding site in the CAT-1 protein of hamsters, thus contributing to resistance of that species. To study the role of receptor glycosylation in animals susceptible to infection, we eliminated loop 3 glycosylation sites by mutagenesis of an mCAT-1 cDNA clone, and we expressed wild-type and mutant receptors in mink fibroblasts and Xenopus oocytes. These receptors had indistinguishable transport properties, as determined by kinetic and voltage-jump electrophysiological studies of arginine uptake in oocytes and by analyses Of L-[3H]arginine uptake in mink cells. Bindings of ecotropic envelope glycoprotein gp7O to the accessible receptor sites on surfaces of mink cells expressing wild-type or mutant mCAT-1 were not significantly different in kinetics or in equilibrium affinities (i.e., K(D) approximately 3.7 X 10(-10) to 7.5 X 10(-10) M). However, when values were normalized to the same levels of mCAT-1 transporter expression, cells with wild-type glycosylated mCAT-1 had only approximately 50% as many sites for gp70 binding as cells with unglycosylated mCAT-1. Although infection with ecotropic MuLV had no effect on activity of the mink CAT-1 transporter that does not bind virus, it caused partial down-modulation of wild-type mCAT-1 and complete down-modulation of unglycosylated mutant mCAT-1. These results suggest that N-linked glycosylation causes wild-type mCAT-1 heterogeneity and that a significant proportion is inaccessible to virus. In part because only the interactive fraction of mCAT-1 can be down-modulated, infected murine cells conserve an amino acid transport capability that supports their viability.

Full Text

The Full Text of this article is available as a PDF (250.0 KB).

Selected References

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

  1. Albritton L. M., Kim J. W., Tseng L., Cunningham J. M. Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses. J Virol. 1993 Apr;67(4):2091–2096. doi: 10.1128/jvi.67.4.2091-2096.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Albritton L. M., Tseng L., Scadden D., Cunningham J. M. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell. 1989 May 19;57(4):659–666. doi: 10.1016/0092-8674(89)90134-7. [DOI] [PubMed] [Google Scholar]
  3. Barchan D., Kachalsky S., Neumann D., Vogel Z., Ovadia M., Kochva E., Fuchs S. How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7717–7721. doi: 10.1073/pnas.89.16.7717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  6. Cone R. D., Mulligan R. C. High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6349–6353. doi: 10.1073/pnas.81.20.6349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eiden M. V., Farrell K., Warsowe J., Mahan L. C., Wilson C. A. Characterization of a naturally occurring ecotropic receptor that does not facilitate entry of all ecotropic murine retroviruses. J Virol. 1993 Jul;67(7):4056–4061. doi: 10.1128/jvi.67.7.4056-4061.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eiden M. V., Farrell K., Wilson C. A. Glycosylation-dependent inactivation of the ecotropic murine leukemia virus receptor. J Virol. 1994 Feb;68(2):626–631. doi: 10.1128/jvi.68.2.626-631.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elbein A. D. Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing. FASEB J. 1991 Dec;5(15):3055–3063. doi: 10.1096/fasebj.5.15.1743438. [DOI] [PubMed] [Google Scholar]
  10. Gazzola G. C., Dall'Asta V., Franchi-Gazzola R., White M. F. The cluster-tray method for rapid measurement of solute fluxes in adherent cultured cells. Anal Biochem. 1981 Aug;115(2):368–374. doi: 10.1016/0003-2697(81)90019-1. [DOI] [PubMed] [Google Scholar]
  11. Gottlieb C., Baenziger J., Kornfeld S. Deficient uridine diphosphate-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity in a clone of Chinese hamster ovary cells with altered surface glycoproteins. J Biol Chem. 1975 May 10;250(9):3303–3309. [PubMed] [Google Scholar]
  12. Handelin B. L., Kabat D. Cell surface receptors for murine leukemia viruses: two assays and their implications. Virology. 1985 Jan 15;140(1):183–187. doi: 10.1016/0042-6822(85)90458-1. [DOI] [PubMed] [Google Scholar]
  13. Harpaz N., Schachter H. Control of glycoprotein synthesis. Processing of asparagine-linked oligosaccharides by one or more rat liver Golgi alpha-D-mannosidases dependent on the prior action of UDP-N-acetylglucosamine: alpha-D-mannoside beta 2-N-acetylglucosaminyltransferase I. J Biol Chem. 1980 May 25;255(10):4894–4902. [PubMed] [Google Scholar]
  14. Kabat D. Cell surface receptors for ecotropic murine retroviruses: mobile membrane proteins that mediate binding and slow endocytosis of the viral envelope glycoprotein. Virology. 1989 Aug;171(2):467–474. doi: 10.1016/0042-6822(89)90616-8. [DOI] [PubMed] [Google Scholar]
  15. Kavanaugh M. P., Wang H., Zhang Z., Zhang W., Wu Y. N., Dechant E., North R. A., Kabat D. Control of cationic amino acid transport and retroviral receptor functions in a membrane protein family. J Biol Chem. 1994 Jun 3;269(22):15445–15450. [PubMed] [Google Scholar]
  16. Kim J. W., Closs E. I., Albritton L. M., Cunningham J. M. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. Nature. 1991 Aug 22;352(6337):725–728. doi: 10.1038/352725a0. [DOI] [PubMed] [Google Scholar]
  17. Kim J. W., Cunningham J. M. N-linked glycosylation of the receptor for murine ecotropic retroviruses is altered in virus-infected cells. J Biol Chem. 1993 Aug 5;268(22):16316–16320. [PubMed] [Google Scholar]
  18. Kleinerman E. S., Lachman L. B., Knowles R. D., Snyderman R., Cianciolo G. J. A synthetic peptide homologous to the envelope proteins of retroviruses inhibits monocyte-mediated killing by inactivating interleukin 1. J Immunol. 1987 Oct 1;139(7):2329–2337. [PubMed] [Google Scholar]
  19. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Masuda M., Remington M. P., Hoffman P. M., Ruscetti S. K. Molecular characterization of a neuropathogenic and nonerythroleukemogenic variant of Friend murine leukemia virus PVC-211. J Virol. 1992 May;66(5):2798–2806. doi: 10.1128/jvi.66.5.2798-2806.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miller A. D., Law M. F., Verma I. M. Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene. Mol Cell Biol. 1985 Mar;5(3):431–437. doi: 10.1128/mcb.5.3.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miller D. G., Miller A. D. Inhibitors of retrovirus infection are secreted by several hamster cell lines and are also present in hamster sera. J Virol. 1993 Sep;67(9):5346–5352. doi: 10.1128/jvi.67.9.5346-5352.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Miller D. G., Miller A. D. Tunicamycin treatment of CHO cells abrogates multiple blocks to retrovirus infection, one of which is due to a secreted inhibitor. J Virol. 1992 Jan;66(1):78–84. doi: 10.1128/jvi.66.1.78-84.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Park B. H., Matuschke B., Lavi E., Gaulton G. N. A point mutation in the env gene of a murine leukemia virus induces syncytium formation and neurologic disease. J Virol. 1994 Nov;68(11):7516–7524. doi: 10.1128/jvi.68.11.7516-7524.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rademacher T. W., Parekh R. B., Dwek R. A. Glycobiology. Annu Rev Biochem. 1988;57:785–838. doi: 10.1146/annurev.bi.57.070188.004033. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Shakin-Eshleman S. H., Spitalnik S. L., Kasturi L. The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency. J Biol Chem. 1996 Mar 15;271(11):6363–6366. doi: 10.1074/jbc.271.11.6363. [DOI] [PubMed] [Google Scholar]
  29. Sitbon M., Sola B., Evans L., Nishio J., Hayes S. F., Nathanson K., Garon C. F., Chesebro B. Hemolytic anemia and erythroleukemia, two distinct pathogenic effects of Friend MuLV: mapping of the effects to different regions of the viral genome. Cell. 1986 Dec 26;47(6):851–859. doi: 10.1016/0092-8674(86)90800-7. [DOI] [PubMed] [Google Scholar]
  30. Stoll J., Wadhwani K. C., Smith Q. R. Identification of the cationic amino acid transporter (System y+) of the rat blood-brain barrier. J Neurochem. 1993 May;60(5):1956–1959. doi: 10.1111/j.1471-4159.1993.tb13428.x. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Talbot S. J., Weiss R. A., Schulz T. F. Reduced glycosylation of human cell lines increases susceptibility to CD4-independent infection by human immunodeficiency virus type 2 (LAV-2/B). J Virol. 1995 Jun;69(6):3399–3406. doi: 10.1128/jvi.69.6.3399-3406.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang H., Dechant E., Kavanaugh M., North R. A., Kabat D. Effects of ecotropic murine retroviruses on the dual-function cell surface receptor/basic amino acid transporter. J Biol Chem. 1992 Nov 25;267(33):23617–23624. [PubMed] [Google Scholar]
  34. Wang H., Kavanaugh M. P., North R. A., Kabat D. Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter. Nature. 1991 Aug 22;352(6337):729–731. doi: 10.1038/352729a0. [DOI] [PubMed] [Google Scholar]
  35. Wang H., Paul R., Burgeson R. E., Keene D. R., Kabat D. Plasma membrane receptors for ecotropic murine retroviruses require a limiting accessory factor. J Virol. 1991 Dec;65(12):6468–6477. doi: 10.1128/jvi.65.12.6468-6477.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilson C. A., Eiden M. V. Viral and cellular factors governing hamster cell infection by murine and gibbon ape leukemia viruses. J Virol. 1991 Nov;65(11):5975–5982. doi: 10.1128/jvi.65.11.5975-5982.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yoshimoto T., Yoshimoto E., Meruelo D. Identification of amino acid residues critical for infection with ecotropic murine leukemia retrovirus. J Virol. 1993 Mar;67(3):1310–1314. doi: 10.1128/jvi.67.3.1310-1314.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES