S2' cleavage site plays a decisive role in expansion of Vero cell tropism by infectious bronchitis virus HV80, with Q855H promoting cell-to-cell fusion

Infectious bronchitis virus (IBV) has restricted cell tropism. Apart from the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered the development of IBV vaccines and research on mechanisms of IBV infection. In a previous study, a new Vero-cell-adapted strain HV80 was obtained via serial chicken embryo and cell passaging of strain H120 and 17 mutations leading to amino acid substitutions occurred in replication gene 1a, S gene and E gene. This study, we constructed recombinants that expressed chimeric S glycoprotein, S1 or S2 subunit of strain H120, and demonstrated that mutations in S2 subunit were related to the Vero cell adaption of strain HV80. With a genome backbone of strain HV80 or H120, and expression of chimeric S2' cleavage site of H120 or HV80, two recombinants demonstrated that the RRRR690/S motif at the S2' cleavage site played a key role in Vero cell adaption of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that F692V enhanced the capability of invasion of HV80 strain, and Q855H induced the formation of syncytia. A transient transfection assay demonstrated different mechanisms for virus-to-cell fusion and cell-to-cell fusion induced by S glycoprotein. The PRRR690/S motif at the S2' cleavage site could be activated by proteases in the process of cell-to-cell fusion, while H855Q substitution did not affect the cell invasion of HV80, but hindered the cell-to-cell fusion by blocking activation of the S2' cleavage site. IMPORTANCEInfectious bronchitis is an acute respiratory disease that has caused large economic losses to the poultry industry. As a member of the gamma-coronaviruses, the restricted cell tropism of infectious bronchitis virus (IBV) limits the development of cellular vaccines and research on infection mechanisms. As a strain that can replicate effectively in mammalian cell lines, studies of HV80s adaptive mechanisms point a way for engineering other IBVs for adaptation in mammalian cell lines. In our study, different recombinants were constructed by reverse genetic techniques, and demonstrated the different mechanism between virus-to-cell and cell-to-cell fusion induced by HV80 S glycoprotein. The acquisition of a highly efficient S2' cleavage site enabled the virus to invade Vero cells. The Q855H substitution played a key role in cell-to-cell fusion, and provided a more efficient model of infection in Vero cells. Our study provides new theoretical insights into mechanisms of IBV adaptation in mammalian cell lines.