National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, People's Republic of China.
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, People's Republic of China.
J Virol. 2023 May 31;97(5):e0048923. doi: 10.1128/jvi.00489-23. Epub 2023 Apr 25.
Infectious bronchitis virus (IBV) infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host factors and fuses the viral and cell membranes. The N-terminal domain of the S1 subunit of IBV S protein binds to sialic acids, but the precise location of the sialic acid binding domain (SABD) and the role of the SABD in IBV-infected chickens remain unclear. Here, we identify the S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19) and GD (GI-7), and the corresponding region of M41 (GI-1), as the minimal SABD using truncated protein histochemistry and neuraminidase assays. Both α-2,3- and α-2,6-linked sialic acids on the surfaces of CEK cells can be used as attachment receptors by IBV, leading to increased infection efficiency. However, 9-O acetylation of the sialic acid glycerol side chain inhibits IBV S1 and SABD protein binding. We further constructed recombinant strains in which the S1 gene or the SABD in the GD and SD genomes were replaced with the corresponding region from M41 by reverse genetics. Infecting chickens with these viruses revealed that the virulence and nephrotropism of rSD, rSD, rGD, and rGD strains were decreased to various degrees compared to their parental strains. A positive sera cross-neutralization test showed that the serotypes were changed for the recombinant viruses. Our results provide insight into IBV infection of host cells that may aid vaccine design. To date, only α-2,3-linked sialic acid has been identified as a potential host binding receptor for IBV. Here, we show the minimum region constituting the sialic acid binding domain (SABD) and the binding characteristics of the S1 subunit of spike (S) protein of IBV strains SD (GI-19), GD (GI-7), and M41 (GI-1) to various sialic acids. The 9-O acetylation modification partially inhibits IBV from binding to sialic acid, while the virus can also bind to sialic acid molecules linked to host cells through an α-2,6 linkage, serving as another receptor determinant. Substitution of the putative SABD from strain M41 into strains SD and GD resulted in reduced virulence, nephrotropism, and a serotype switch. These findings suggest that sialic acid binding has diversified during the evolution of γ-coronaviruses, impacting the biological properties of IBV strains. Our results offer insight into the mechanisms by which IBV invades host cells.
传染性支气管炎病毒（IBV）感染是由跨膜刺突（S）糖蛋白引发的，该蛋白与宿主因子结合并融合病毒和细胞膜。IBV S 蛋白 S1 亚基的 N 端结构域与唾液酸结合，但 IBV 感染鸡中唾液酸结合域（SABD）的确切位置和 SABD 的作用仍不清楚。在这里，我们使用截短蛋白组织化学和神经氨酸酶测定法鉴定了 IBV 株 SD（GI-19）和 GD（GI-7）的 S1 N 端氨基酸（aa）残基 19 至 227（共 209 个 aa），以及 M41（GI-1）的相应区域，作为最小的 SABD。CEK 细胞表面的α-2,3-和α-2,6 连接的唾液酸都可以被 IBV 用作附着受体，从而提高感染效率。然而，唾液酸甘油侧链的 9-O 乙酰化抑制了 IBV S1 和 SABD 蛋白的结合。我们进一步通过反向遗传学构建了重组株，其中 GD 和 SD 基因组中的 S1 基因或 SABD 被 M41 的相应区域取代。用这些病毒感染鸡发现，与亲本株相比，rSD、rSD、rGD 和 rGD 株的毒力和肾嗜性均不同程度降低。阳性血清交叉中和试验表明重组病毒的血清型发生了变化。我们的结果提供了 IBV 感染宿主细胞的见解，这可能有助于疫苗设计。迄今为止，只有α-2,3 连接的唾液酸被鉴定为 IBV 的潜在宿主结合受体。在这里，我们展示了 SD（GI-19）、GD（GI-7）和 M41（GI-1）株的 S 蛋白刺突（S）的 S1 亚基构成唾液酸结合域（SABD）的最小区域和结合特征各种唾液酸。9-O 乙酰化修饰部分抑制了 IBV 与唾液酸的结合，而病毒也可以与通过α-2,6 键连接到宿主细胞的唾液酸分子结合，作为另一个受体决定簇。将来自 M41 株的假定 SABD 替换为 SD 和 GD 株导致毒力、肾嗜性降低和血清型转变。这些发现表明，在γ冠状病毒的进化过程中，唾液酸结合已经多样化，影响了 IBV 株的生物学特性。我们的研究结果为 IBV 入侵宿主细胞的机制提供了深入了解。
