Identification of N-linked glycosylation sites in the spike protein and their functional impact on the replication and infectivity of coronavirus infectious bronchitis virus in cell culture

•Among 29 putative glycosylation sites in the IBV spike (S) protein, 8 sites are confirmed by mass spectrometry analysis.•N-D/Q mutations at N212 and N276 abolish the fusion activity of IBV S protein and the infectivity of recombinant viruses.•Mutations at other glycosylation sites differentially affect cleavage and fusion of IBV S protein, and infectivity of rIBVs.•N283 in IBV S protein is critically involved in IBV replication and infectivity independent of N-linked glycosylation.

Spike (S) glycoprotein on the viral envelope is the main determinant of infectivity. The S protein of coronavirus infectious bronchitis virus (IBV) contains 29 putative asparagine(N)-linked glycosylation sites. These post-translational modifications may assist in protein folding and play important roles in the functionality of S protein. In this study, we used bioinformatics tools to predict N-linked glycosylation sites and to analyze their distribution in IBV strains and variants. Among these sites, 8 sites were confirmed in the S protein extracted from partially purified virus particles by proteomics approaches. N-D and N-Q substitutions at 13 predicted sites were introduced into an infectious clone system. The impact on S protein-mediated cell-cell fusion, viral recovery and infectivity was assessed, leading to the identification of sites essential for the functions of IBV S protein. Further characterization of these and other uncharacterized sites may reveal novel aspects of N-linked glycosylation in coronavirus replication and pathogenesis.