Unique amino acid substitution within the spike glycoprotein S1 subunit promotes infectious bronchitis virus infection via enhancing viral attachment
- 1Division of Avian Infectious Diseases, State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China
Abstract
Due to the scarcity of cell-adapted strains and limitations of current reverse genetics techniques, the factors determining cell tropism of infectious bronchitis virus (IBV), a prototype gammacoronavirus, remain unclear. Here, we demonstrated that the expanded cell tropism of CEA, a chicken embryo fibroblast-adapted strain derived from tl/CH/LDT3/03, was related to its enhanced attachment capacity. Then, a reverse genetics platform for IBV based on circular polymerase extension reaction (CPER) was established, enabling the successful recovery of rCEA and rLDT, which retained the biological properties of their respective parental viruses. Using this platform, we constructed a series of chimeric viruses based on the CEA strain by incorporating the entire S gene, the S1 and S2 subunits, and substitutions at differential sites derived from the tl/CH/LDT3/03 strain. Our findings indicated that subunit S1, rather than S2, was associated with viral adaptation in DF-1 cells, with the amino acid residue at position 413 in the C-terminal domain of the S1 protein as a key determinant. Evaluation with recombinant S1 proteins and chimeric viruses revealed that the histidine residue at position 413 (His413) promoted enhanced attachment of both S1 proteins and viruses to cells, while exerting no direct effect on viral entry. Mechanistically, His413 facilitated efficient viral binding to α-2,3-linked sialic acids presented on gangliosides rather than glycoproteins. This interaction activated Src kinase and triggered caveolae-mediated endocytosis, initiating viral entry and subsequent replication. Collectively, the CPER-based reverse genetics platform established in this study represents a significant technical advancement for IBV, and our findings provide novel insights into the cell adaptation and entry mechanisms of IBV.
IMPORTANCE: Although infectious bronchitis virus (IBV) was the first discovered coronavirus, the cellular receptors and cofactors that mediate successful infection, as well as the specific pathways and mechanisms of viral entry into host cells, remain to be elucidated. In this study, we established a reverse genetics platform for IBV using circular polymerase extension reaction for the first time. A series of chimeric viruses were subsequently recovered, and a specific amino acid substitution at position 413 in the C-terminal domain of the S1 subunit was identified as a key determinant for the expanded DF-1 cell tropism of IBV. The histidine residue at this site facilitated viral binding to α-2,3-linked sialic acids on gangliosides, thereby activating the caveola-mediated endocytosis and enabling viral entry into DF-1 cells. This study provides novel insights into the receptor-binding function of the IBV S protein and the strategies employed by the virus for cell adaptation. Additionally, our findings offer new perspectives for developing cell-culture-based vaccines.
Related
- Molecular epizootiology of avian infectious bronchitis in Russia
- Phylogenetic analysis of partial S1 and N gene sequences of infectious bronchitis virus isolates from Italy revealed genetic diversity and recombination
- Preliminary association between infectious bronchitis virus viral load and the tracheal microbiome of broiler chickens
- Molecular characterization of infectious bronchitis virus isolates from Russia and neighbouring countries: identification of intertypic recombination in the S1 gene
- Detection of Avian Infectious Bronchitis Virus Genotype GI-13 in the Almaty Region, Kazakhstan
BibTeX
@article{liu2026unique,
title = {{Unique amino acid substitution within the spike glycoprotein S1 subunit promotes infectious bronchitis virus infection via enhancing viral attachment}},
author = {Tianyi Liu and Yujuan Zhang and Zongxi Han and Huixin Li and Yuhao Shao and Junfeng Sun and Shengwang Liu},
journal = {mBio},
year = {2026},
month = sep,
doi = {10.1128/mbio.00884-26},
url = {https://doi.org/10.1128/mbio.00884-26},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Unique amino acid substitution within the spike glycoprotein S1 subunit promotes infectious bronchitis virus infection via enhancing viral attachment
AU - Tianyi Liu
AU - Yujuan Zhang
AU - Zongxi Han
AU - Huixin Li
AU - Yuhao Shao
AU - Junfeng Sun
AU - Shengwang Liu
PY - 2026
DA - 2026/09/03
JO - mBio
DO - 10.1128/mbio.00884-26
UR - https://doi.org/10.1128/mbio.00884-26
AB - Due to the scarcity of cell-adapted strains and limitations of current reverse genetics techniques, the factors determining cell tropism of infectious bronchitis virus (IBV), a prototype gammacoronavirus, remain unclear. Here, we demonstrated that the expanded cell tropism of CEA, a chicken embryo fibroblast-adapted strain derived from tl/CH/LDT3/03, was related to its enhanced attachment capacity. Then, a reverse genetics platform for IBV based on circular polymerase extension reaction (CPER) was established, enabling the successful recovery of rCEA and rLDT, which retained the biological properties of their respective parental viruses. Using this platform, we constructed a series of chimeric viruses based on the CEA strain by incorporating the entire S gene, the S1 and S2 subunits, and substitutions at differential sites derived from the tl/CH/LDT3/03 strain. Our findings indicated that subunit S1, rather than S2, was associated with viral adaptation in DF-1 cells, with the amino acid residue at position 413 in the C-terminal domain of the S1 protein as a key determinant. Evaluation with recombinant S1 proteins and chimeric viruses revealed that the histidine residue at position 413 (His413) promoted enhanced attachment of both S1 proteins and viruses to cells, while exerting no direct effect on viral entry. Mechanistically, His413 facilitated efficient viral binding to α-2,3-linked sialic acids presented on gangliosides rather than glycoproteins. This interaction activated Src kinase and triggered caveolae-mediated endocytosis, initiating viral entry and subsequent replication. Collectively, the CPER-based reverse genetics platform established in this study represents a significant technical advancement for IBV, and our findings provide novel insights into the cell adaptation and entry mechanisms of IBV. IMPORTANCE: Although infectious bronchitis virus (IBV) was the first discovered coronavirus, the cellular receptors and cofactors that mediate successful infection, as well as the specific pathways and mechanisms of viral entry into host cells, remain to be elucidated. In this study, we established a reverse genetics platform for IBV using circular polymerase extension reaction for the first time. A series of chimeric viruses were subsequently recovered, and a specific amino acid substitution at position 413 in the C-terminal domain of the S1 subunit was identified as a key determinant for the expanded DF-1 cell tropism of IBV. The histidine residue at this site facilitated viral binding to α-2,3-linked sialic acids on gangliosides, thereby activating the caveola-mediated endocytosis and enabling viral entry into DF-1 cells. This study provides novel insights into the receptor-binding function of the IBV S protein and the strategies employed by the virus for cell adaptation. Additionally, our findings offer new perspectives for developing cell-culture-based vaccines.
SN - 2150-7511
ER -