Disruption of the S1/S2 multibasic cleavage site attenuates infectious bronchitis virus, while S2′ partially restores viral virulence and expands tissue tropism

Multibasic cleavage motifs play critical roles in envelope glycoprotein-mediated viral entry by serving as substrates for proprotein convertases like furin. The gamma-coronavirus infectious bronchitis virus (IBV) inherently possesses a conserved S1/S2 multibasic cleavage site (MBCS) within its S gene. Notably, the IBV Beaudette strain uniquely harbors an additional S2′ MBCS, which extends its cellular tropism. Our previous work demonstrated that introducing the S2′ MBCS into the YN strain contributes to central nervous system damage. However, the functional interplay between the S1/S2 and S2′ MBCS has not yet been elucidated. Herein, we rescued several recombinant IBV mutants based on two different genotypes, engineered with distinct S protein cleavage patterns. Our results revealed that deletion of the S1/S2 MBCS results in a drastic reduction of viral replication efficiency and pathogenicity. This negative effect can be partially compensated by introducing the S2′ MBCS. The S2′ MBCS also rendered increased mortality and elevated viral loads in neurological organs—effects that were diminished with less efficient S1/S2 cleavage. Inconsistencies exist regarding the impact of MBCS mutations in different IBV strain backgrounds, which are mainly due to their differential dependence on host proteases, correlating with their distinct tissue tropism. Our study demonstrated a proteolytic coordination model for IBV S protein activation: The S1/S2 MBCS primarily confers efficient replication competence; the S2′ site acts as an amplifier of organ-specific pathogenicity, particularly enhancing neurotropism. This synergistic relationship is conserved across different IBV strains. IMPORTANCE Multibasic cleavage sites (MBCSs) are critical determinants of coronavirus pathogenicity and tissue tropism. While infectious bronchitis virus (IBV) inherently possesses a conserved S1/S2 cleavage site, the functional significance of this site and its potential interplay with the unique S2′ MBCS found in the Beaudette strain remain poorly understood. Our study definitively establishes the critical role of the S1/S2 cleavage site for efficient IBV replication in vitro and full virulence in vivo , while the S2′ site can only partially restore viral virulence. The pathogenic enhancement conferred by the S2′ site is due to the heightened viral loads and enhanced organ-specific neurological damage. These findings provide essential insights into IBV pathogenesis governed by spike protein cleavage and establish a vital theoretical foundation for designing rationally attenuated vaccines and effective control strategies against avian coronaviruses.