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.

病毒包膜上的穗状糖蛋白是感染性的主要决定因素。冠状病毒感染性支气管炎病毒（IBV）的S蛋白包含29个假定的天冬酰胺（N）连接的糖基化位点。这些翻译后修饰可协助蛋白质折叠并在S蛋白质的功能中起重要作用。在这项研究中，我们使用生物信息学工具预测了N-连接的糖基化位点，并分析了它们在IBV株和变种中的分布。在这些位点中，通过蛋白质组学方法从部分纯化的病毒颗粒提取的S蛋白中确认了8个位点。将13个预测位点的ND和NQ替代引入感染性克隆系统。评估了对S蛋白介导的细胞-细胞融合，病毒恢复和感染性的影响，导致鉴定出IBV S蛋白功能必不可少的位点。这些和其他未表征位点的进一步表征可能揭示冠状病毒复制和发病机理中N-联糖基化的新方面。