Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein.
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.
Hu
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Microbiology and Tumorbiology Centre, Karolinska Institute, Stockholm, Sweden.
Cathomen
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Cattaneo
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Norrby
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D000595:Amino Acid Sequence; D001216:Asparagine / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000821:virology; D004721:Endoplasmic Reticulum / Q000821:virology; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D008459:Measles virus / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D009844:Oligosaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics; D014760:Viral Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D009844:Oligosaccharides; D021382:Protein Sorting Signals; D014760:Viral Fusion Proteins; D001216:Asparagine