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

Hao Wang, Elizabeth Klamo, Shawn E. Kuhmann, Susan L. Kozak, Michael P. Kavanaugh, David Kabat

Research output: Contribution to journalArticle

34 Citations (Scopus)

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 gp70 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) ≃ 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.

Original languageEnglish (US)
Pages (from-to)6884-6891
Number of pages8
JournalJournal of Virology
Volume70
Issue number10
StatePublished - Oct 1996

Fingerprint

Retroviridae
amino acid transporters
Amino Acid Transport Systems
Cell Surface Receptors
glycosylation
Glycosylation
receptors
mice
Mink
Virus Attachment
cells
Murine Leukemia Viruses
mink
Murine leukemia virus
viruses
transporters
Binding Sites
Viruses
Infection
binding sites

ASJC Scopus subject areas

  • Immunology

Cite this

Wang, H., Klamo, E., Kuhmann, S. E., Kozak, S. L., Kavanaugh, M. P., & Kabat, D. (1996). Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter. Journal of Virology, 70(10), 6884-6891.

Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter. / Wang, Hao; Klamo, Elizabeth; Kuhmann, Shawn E.; Kozak, Susan L.; Kavanaugh, Michael P.; Kabat, David.

In: Journal of Virology, Vol. 70, No. 10, 10.1996, p. 6884-6891.

Research output: Contribution to journalArticle

Wang, H, Klamo, E, Kuhmann, SE, Kozak, SL, Kavanaugh, MP & Kabat, D 1996, 'Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter', Journal of Virology, vol. 70, no. 10, pp. 6884-6891.
Wang, Hao ; Klamo, Elizabeth ; Kuhmann, Shawn E. ; Kozak, Susan L. ; Kavanaugh, Michael P. ; Kabat, David. / Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter. In: Journal of Virology. 1996 ; Vol. 70, No. 10. pp. 6884-6891.
@article{573504aa5d5047c082ee8aa753db10bb,
title = "Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter",
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 gp70 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) ≃ 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.",
author = "Hao Wang and Elizabeth Klamo and Kuhmann, {Shawn E.} and Kozak, {Susan L.} and Kavanaugh, {Michael P.} and David Kabat",
year = "1996",
month = "10",
language = "English (US)",
volume = "70",
pages = "6884--6891",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "10",

}

TY - JOUR

T1 - Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter

AU - Wang, Hao

AU - Klamo, Elizabeth

AU - Kuhmann, Shawn E.

AU - Kozak, Susan L.

AU - Kavanaugh, Michael P.

AU - Kabat, David

PY - 1996/10

Y1 - 1996/10

N2 - 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 gp70 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) ≃ 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.

AB - 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 gp70 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) ≃ 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.

UR - http://www.scopus.com/inward/record.url?scp=0029820856&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029820856&partnerID=8YFLogxK

M3 - Article

VL - 70

SP - 6884

EP - 6891

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 10

ER -