Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein’s surface. Here we used IBV propagated in embryonated hens’ eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.

中文翻译：

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对禽冠状病毒刺突蛋白的分析揭示了存在的聚糖的异质性

传染性支气管炎病毒 (IBV) 是一种具有重要经济意义的冠状病毒，作为传染性支气管炎的病原体，给全世界的家禽业造成了破坏性的损失。冠状病毒刺突 (S) 糖蛋白是一种从病毒体表面突出的大型 I 型膜蛋白，有助于附着和进入宿主细胞。IBV S 蛋白被切割成两个亚基，S1 和 S2，后者已被确定为细胞趋向性的决定因素。最近在哺乳动物和昆虫细胞中表达冠状病毒 S 蛋白的研究已经确定了蛋白质表面的高水平糖基化。在这里，我们使用在鸡胚中繁殖的 IBV 来探索源自天然宿主鸡细胞感染的病毒的聚糖谱。在体外，在复合糖基化的化学抑制后对病毒复制没有影响。单克隆或多克隆抗体对病毒的中和作用不受影响。在分析了四种 IBV 菌株的 S 蛋白的预测糖基化位点后，我们通过质谱法确认了实验室致病菌株 M41-CK 的 18 个位点的糖基化。进一步的表征揭示了存在于其中六个位点的聚糖之间的异质性，表明 IBV 病毒体上单个 S 蛋白的聚糖谱存在差异。这些结果证明了复杂聚糖在 IBV 复制中的非特异性作用，表明参与抗体识别但不参与中和。