Yamada Yoshiyuki, Liu Xiao Bo, Fang Shou Guo, Tay Felicia P L, Liu Ding Xiang
Institute of Molecular and Cell Biology, Proteos, Singapore.
PLoS One. 2009 Jul 2;4(7):e6130. doi: 10.1371/journal.pone.0006130.
Coronavirus host and cell specificities are determined by specific interactions between the viral spike (S) protein and host cell receptor(s). Avian coronavirus infectious bronchitis (IBV) has been adapted to embryonated chicken eggs, primary chicken kidney (CK) cells, monkey kidney cell line Vero, and other human and animal cells. Here we report that acquisition of the cell-cell fusion activity by amino acid mutations in the S protein determines the infectivity of IBV in cultured cells. Expression of S protein derived from Vero- and CK-adapted strains showed efficient induction of membrane fusion. However, expression of S protein cloned from the third passage of IBV in chicken embryo (EP3) did not show apparent syncytia formation. By construction of chimeric S constructs and site-directed mutagenesis, a point mutation (L857-F) at amino acid position 857 in the heptad repeat 1 region of S protein was shown to be responsible for its acquisition of the cell-cell fusion activity. Furthermore, a G405-D point mutation in the S1 domain, which was acquired during further propagation of Vero-adapted IBV in Vero cells, could enhance the cell-cell fusion activity of the protein. Re-introduction of L857 back to the S gene of Vero-adapted IBV allowed recovery of variants that contain the introduced L857. However, compensatory mutations in S1 and some distant regions of S2 were required for restoration of the cell-cell fusion activity of S protein carrying L857 and for the infectivity of the recovered variants in cultured cells. This study demonstrates that acquisition of the cell-cell fusion activity in S protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins.
冠状病毒的宿主和细胞特异性由病毒刺突（S）蛋白与宿主细胞受体之间的特定相互作用决定。禽冠状病毒传染性支气管炎病毒（IBV）已适应于鸡胚、原代鸡肾（CK）细胞、猴肾细胞系Vero以及其他人和动物细胞。在此我们报告，S蛋白中的氨基酸突变获得细胞 - 细胞融合活性决定了IBV在培养细胞中的感染性。源自Vero和CK适应株的S蛋白表达显示出高效的膜融合诱导作用。然而，从鸡胚中传代三次的IBV（EP3）克隆的S蛋白表达未显示出明显的多核体形成。通过构建嵌合S构建体和定点诱变，发现S蛋白七肽重复序列1区域氨基酸位置857处的点突变（L857 - F）导致其获得细胞 - 细胞融合活性。此外，在Vero适应的IBV在Vero细胞中进一步传代过程中获得的S1结构域中的G405 - D点突变可增强该蛋白的细胞 - 细胞融合活性。将L857重新引入Vero适应的IBV的S基因可使含有引入的L857的变体恢复。然而，对于携带L857的S蛋白的细胞 - 细胞融合活性的恢复以及所恢复变体在培养细胞中的感染性，S1和S2一些远端区域的补偿性突变是必需的。本研究表明，S蛋白中细胞 - 细胞融合活性的获得决定了冠状病毒从鸡胚到人和动物来源培养细胞的选择和/或适应。
