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 o f the virion envelope, contains four potential sites for attachment of N-linked oligosaccharides. Three are located in the F-2 subunit of th e 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 wil d-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 po lymerase system. Analysis of these proteins revealed that three (resid ues 29, 61 and 67) potential sites for addition of N-linked glycans in the F-2 subunit are actually utilized. The functional glycosylation s ites were systematically removed in all possible combinations from the F protein to form a panel of mutants from which the role of carbohydr ates, singly or in various combinations, could be evaluated. One singl e-site mutant protein lacking the glycosylation site of Asn-67 was pro cessed, transported to the cell surface and could induce cell fusion. However, the other two single-site mutant proteins with deletions of g lycosylation 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 site s resulted in protein retention in the endoplasmic reticulum. Therefor e, it appears that glycosylation of sites Asn-29 and Asn-61 has import ant roles in maintaining the native structure of the F protein.