The
membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN, family Bunyaviridae) contain three potential sites for the attachment of N-linked
glycans: one site (N60) on Gn and two (N624 and N1169) on Gc. We determined that all three sites are glycosylated. Digestion of the
glycoproteins with
endo-beta-N-acetylglucosaminidase H (endo H) or
peptide:N-glycosidase F revealed that Gn and Gc differ significantly in their
glycan status and that late in
infection Gc
glycans remain endo H sensitive. The roles of the N-
glycans in intracellular trafficking of the
glycoproteins to the Golgi, protein folding, and virus replication were investigated by mutational analysis and confocal immunofluorescence. Elimination of the
glycan on Gn, by changing N60 to a Q residue, resulted in the
protein misfolding and failure of both Gn and Gc
proteins to traffic to the Golgi complex. We were unable to rescue a viable virus by reverse genetics from a
cDNA containing the N60Q mutation. In contrast, mutant Gc
proteins lacking
glycans on either N624 or N1169, or both sites, were able to target to the Golgi. Gc
proteins containing mutations N624Q and N1169Q acquired endo H resistance. Three viable N glycosylation-site-deficient viruses, lacking
glycans on one site or both sites on Gc, were created by reverse genetics. The viability of these recombinant viruses and analysis of growth kinetics indicates that the
glycans on Gc are not essential for BUN replication, but they do contribute to the efficiency of
virus infection.