Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein

A. Hu, T. Cathomen, Roberto Cattaneo, E. Norrby

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

The fusion (F) glycoprotein of measles virus, a structural component of the virion envelope, contains four potential sites for attachment of N-linked oligosaccharides. Three are located in the F2 subunit of the protein and one in the signal peptide. Four mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Ser/Thr to Ser-X-Ser/Thr. The wild-type and altered forms of the F protein were expressed in BHK-21 and HeLa T4 cells by use of the recombinant vaccinia virus-encoding T7 polymerase system. Analysis of these proteins revealed that three (residues 29, 61 and 67) potential sites for addition of N-linked glycans in the F2 subunit are actually utilized. The functional glycosylation sites were systematically removed in all possible combinations from the F protein to form a panel of mutants from which the role of carbohydrates, singly or in various combinations, could be evaluated. One single-site mutant protein lacking the glycosylation site of Asn-67 was processed, transported to the cell surface and could induce cell fusion. However, the other two single-site mutant proteins with deletions of glycosylation sites Asn-29 or Asn-61 exhibited a defect in processing, were not transported to cell surface and thus induced no cell fusion. The absence of any two of the three or of all three glycosylation sites resulted in protein retention in the endoplasmic reticulum. Therefore, it appears that glycosylation of sites Asn-29 and Asn-61 has important roles in maintaining the native structure of the F protein.

Original languageEnglish (US)
Pages (from-to)705-710
Number of pages6
JournalJournal of General Virology
Volume76
Issue number3
StatePublished - 1995
Externally publishedYes

Fingerprint

Viral Fusion Proteins
Measles virus
Oligosaccharides
Glycosylation
Cell Fusion
Mutant Proteins
Proteins
CD4-Positive T-Lymphocytes
Vaccinia virus
Protein Subunits
Protein Sorting Signals
Site-Directed Mutagenesis
HeLa Cells
Endoplasmic Reticulum
Virion
Polysaccharides
Glycoproteins
Carbohydrates

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein. / Hu, A.; Cathomen, T.; Cattaneo, Roberto; Norrby, E.

In: Journal of General Virology, Vol. 76, No. 3, 1995, p. 705-710.

Research output: Contribution to journalArticle

@article{4fc01cb8717842ef871cf2a9f685b783,
title = "Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein",
abstract = "The fusion (F) glycoprotein of measles virus, a structural component of the virion envelope, contains four potential sites for attachment of N-linked oligosaccharides. Three are located in the F2 subunit of the protein and one in the signal peptide. Four mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Ser/Thr to Ser-X-Ser/Thr. The wild-type and altered forms of the F protein were expressed in BHK-21 and HeLa T4 cells by use of the recombinant vaccinia virus-encoding T7 polymerase system. Analysis of these proteins revealed that three (residues 29, 61 and 67) potential sites for addition of N-linked glycans in the F2 subunit are actually utilized. The functional glycosylation sites were systematically removed in all possible combinations from the F protein to form a panel of mutants from which the role of carbohydrates, singly or in various combinations, could be evaluated. One single-site mutant protein lacking the glycosylation site of Asn-67 was processed, transported to the cell surface and could induce cell fusion. However, the other two single-site mutant proteins with deletions of glycosylation sites Asn-29 or Asn-61 exhibited a defect in processing, were not transported to cell surface and thus induced no cell fusion. The absence of any two of the three or of all three glycosylation sites resulted in protein retention in the endoplasmic reticulum. Therefore, it appears that glycosylation of sites Asn-29 and Asn-61 has important roles in maintaining the native structure of the F protein.",
author = "A. Hu and T. Cathomen and Roberto Cattaneo and E. Norrby",
year = "1995",
language = "English (US)",
volume = "76",
pages = "705--710",
journal = "Journal of General Virology",
issn = "0022-1317",
publisher = "Society for General Microbiology",
number = "3",

}

TY - JOUR

T1 - Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein

AU - Hu, A.

AU - Cathomen, T.

AU - Cattaneo, Roberto

AU - Norrby, E.

PY - 1995

Y1 - 1995

N2 - The fusion (F) glycoprotein of measles virus, a structural component of the virion envelope, contains four potential sites for attachment of N-linked oligosaccharides. Three are located in the F2 subunit of the protein and one in the signal peptide. Four mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Ser/Thr to Ser-X-Ser/Thr. The wild-type and altered forms of the F protein were expressed in BHK-21 and HeLa T4 cells by use of the recombinant vaccinia virus-encoding T7 polymerase system. Analysis of these proteins revealed that three (residues 29, 61 and 67) potential sites for addition of N-linked glycans in the F2 subunit are actually utilized. The functional glycosylation sites were systematically removed in all possible combinations from the F protein to form a panel of mutants from which the role of carbohydrates, singly or in various combinations, could be evaluated. One single-site mutant protein lacking the glycosylation site of Asn-67 was processed, transported to the cell surface and could induce cell fusion. However, the other two single-site mutant proteins with deletions of glycosylation sites Asn-29 or Asn-61 exhibited a defect in processing, were not transported to cell surface and thus induced no cell fusion. The absence of any two of the three or of all three glycosylation sites resulted in protein retention in the endoplasmic reticulum. Therefore, it appears that glycosylation of sites Asn-29 and Asn-61 has important roles in maintaining the native structure of the F protein.

AB - The fusion (F) glycoprotein of measles virus, a structural component of the virion envelope, contains four potential sites for attachment of N-linked oligosaccharides. Three are located in the F2 subunit of the protein and one in the signal peptide. Four mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Ser/Thr to Ser-X-Ser/Thr. The wild-type and altered forms of the F protein were expressed in BHK-21 and HeLa T4 cells by use of the recombinant vaccinia virus-encoding T7 polymerase system. Analysis of these proteins revealed that three (residues 29, 61 and 67) potential sites for addition of N-linked glycans in the F2 subunit are actually utilized. The functional glycosylation sites were systematically removed in all possible combinations from the F protein to form a panel of mutants from which the role of carbohydrates, singly or in various combinations, could be evaluated. One single-site mutant protein lacking the glycosylation site of Asn-67 was processed, transported to the cell surface and could induce cell fusion. However, the other two single-site mutant proteins with deletions of glycosylation sites Asn-29 or Asn-61 exhibited a defect in processing, were not transported to cell surface and thus induced no cell fusion. The absence of any two of the three or of all three glycosylation sites resulted in protein retention in the endoplasmic reticulum. Therefore, it appears that glycosylation of sites Asn-29 and Asn-61 has important roles in maintaining the native structure of the F protein.

UR - http://www.scopus.com/inward/record.url?scp=0028899494&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028899494&partnerID=8YFLogxK

M3 - Article

VL - 76

SP - 705

EP - 710

JO - Journal of General Virology

JF - Journal of General Virology

SN - 0022-1317

IS - 3

ER -