THE ROLE OF INDIVIDUAL OLIGOSACCHARIDE CHAINS IN THE ACTIVITIES OF THE HN GLYCOPROTEIN OF NEWCASTLE-DISEASE VIRUS

To explore the role of N-linked carbohydrate in the activities of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV), the six glycosylation addition sites (G1-G6) in the HN sequence of the AV strain of NDV were mutated. Migration of mutant protein on polyacrylamide gels as well as endoglycosidase H digestion of mutant protein showed that four of the addition sites (G1, G2, G3, and G4 at amino acids 119, 341, 433, and 481, respectively) are used while two (G5 and G6 at amino acids 508 and 538, respectively) are not used. Proteins expressed from single and all possible combinations of double and triple mutant DNAs as well as the unglycosylated molecule were characterized for the presence of specific antigenic sites, formation of disulfide-linked dimers, stability, transport to the cell surface, and biological activity. Results showed that glycosylation al positions G1 and G2 play little detectable role in the folding, stability, or transport of the molecule either singly or in combination with other mutations. Mutation of these sequons, however, significantly increased the cell attachment and fusion promotion activities of the protein, particularly in combination. Mutation of the glycosylation site at G4 either singly or in combination with other site-eliminating mutants inhibited the formation of the mature protein, while a mutation eliminating the addition site at G3 had a slight effect on the efficiency of folding, particularly in combination with mutation of the site G4. When normalized to surface expression, elimination of carbohydrate addition sites at G3 and G4 singly or in combination with other mutations depressed in particular the neuraminidase activity of the protein but not the fusion promotion activity. Thus two oligosaccharides do not have a detectable role in maturation but do modulate the biological activities of the protein. The other two oligosaccharides influence both folding and activity Of the protein. (C) 1995 Academic Press, Inc.