FUNCTIONAL-ANALYSIS OF N-LINKED GLYCOSYLATION MUTANTS OF THE MEASLES-VIRUS FUSION PROTEIN SYNTHESIZED BY RECOMBINANT VACCINIA VIRUS VECTORS

The role of N-linked glycosylation in the biological activity of the m easles virus (MV) fusion (F) protein was analyzed by expressing glycos ylation mutants with recombinant vaccinia virus vectors. There are thr ee potential N-linked glycosylation sites located on the F, subunit po lypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant protei ns was reduced by 50% relative to the wild-type level when glycosylati on at either Asn-29 or Asn-61 was abolished. Despite the similar level s of cell surface expression, the Asn-29 and Asn-61 mutant proteins ha d different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not ex hibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant prot ein but had little effect on its ability to cause cell fusion. However , when the Asn-67 mutation was combined with mutations at either of th e other two sites, cleavage-dependent activation, cell surface express ion, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the prot eolytic cleavage, stability, and biological activity of the MV F prote in. The oligosaccharide side chains in MV F are thus essential for opt imum conformation of the extracellular F-2 subunit that is presumed to bind cellular membranes.