Biosynthesis of the oligosaccharides of influenza viral glycoproteins.

Glycosylation of influenza viral glycoproteins was investigated by pulse-labeling of infected BHK21-F cells with radioactive sugar precursors and by cell fractionation and analysis of Pronase-digested viral glycopeptides by gel filtration. The results with short pulses of [3H]mannose suggested that the initial event in glycosylation is the en bloc transfer of oligomannosyl cores to viral glycoproteins associated with rough membranes. The molecular weight of the glycopeptides which represent the cores was estimated to be approximately 1600-2200. Some mannose residues appear to be subsequently removed from oligosaccharide cores. [3H]mannose-labeled glycopeptides obtained either from cells pulsed for brief periods or from rough membranes, which contain predominantly oligosaccharide cores, were sensitive to digestion by endo-p-N-acetylglucosaminidase H (endo-H). On the other hand, glycopeptides larger than oligosaccharide cores, which appeared during chases or after migration of viral glycoproteins from rough to smooth membranes, were resistant to endo-H treatment. The branched sugars (glucosamine, galactose, and fucose), which are contained only in the complex (type I) oligosaccharide chains of virions, appear to be added in a stepwise manner to the trimmed oligosaccharide cores primarily on smooth membranes. Mannoserich glycopeptides of virions (type II) are similar in size to oligosaccharide cores detected in infected cells and are totally sensitive to endo-H, suggesting that type II glycopeptides may represent oligomannosyl cores which escape trimming as well as addition of branched sugars. Comparison of glycopeptides of infected and uninfected BHK21-F cells suggests that influenza viral glycoproteins contain oligosaccharide chains similar in size to those of host cells except for the absence of sialic acid in viral glycoproteins. Further, we observed that intracytoplasmic membranes from infected cells contain much less sialic acid than those from uninfected cells, indicating that viral neuraminidase present in the interior of infected cells possesses enzymatic activity.