Monosaccharide Sequence of Protein-Bound Glycans of Uukuniemi Virus

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Abstract
Uukuniemi virus, a member of the Bunyaviridae family, was grown in hamster kidney BHK-21 cells in the presence of [3H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [3H]mannose-labeled glycopeptides of the mixture of the 2 envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo- and endoglycosidases, by chemical methods and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into 3 categories: complex, high-mannose type and intermediate. The complex glycopeptides probably contained mainly 2 NeuNAc-Gal-GlcNAc branches attached to a core (Man)3(GlcNAc)2 peptide. The high-mannose-type glycans were estimated to contain at least 5 mannose units attached to 2 N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The intermediate glycopeptides apparently contain a mannosyl core. In about half of the molecules, 1 branch appeared to be terminated in mannose and 1 appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. EM data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [3H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62 and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [35S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.