The effects of -linked oligosaccharides on the haemagglutinin (H) protein of measles virus (MV) were assessed with respect to the processing and antigenicity of the molecule. The functional glycosylation sites on the H protein were determined by eliminating each of the five potential positions, Asn-168, Asn-187, Asn-200, Asn-215 and Asn-238, for -linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant H proteins in BHK-21 cells by a recombinant vaccinia virus encoding T7 polymerase indicated that the first four sites were used in the H glycoprotein for the addition of -linked oligosaccharide chains. Heterogeneity of oligosaccharide processing was demonstrated. One of the four glycosylation sites had a different carbohydrate structure from those of the other three glycosylation sites and this varied glycosylation was responsible for the appearance of two forms of the H protein. The functional glycosylation sites were systematically removed in various combinations from the H protein to form a panel of mutants in which the role of carbohydrate chains, singly or in different combinations, could be evaluated. Investigations of these glycosylation mutants indicated that (i) two of the four individual carbohydrate side-chains have a large influence on the antigenicity of the molecule; (ii) individual carbohydrate side-chains have little effect on the folding and oligomerization of the molecule, and are not sufficient or necessary alone to facilitate the transport of the molecule to the plasma membrane; (iii) at least two carbohydrate side-chains are required for the H protein to move along the exocytic pathway to the plasma membrane and various combinations of oligosaccharide side-chains, irrespective of the carbohydrate localizations, influence equally the processing of the molecule.


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