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Coronaviruses have evolved a variety of strategies to optimize cellular microenvironment for efficient replication. In this study, we report the induction of AP-1 transcription factors by coronavirus infection based on genome-wide analyses of differentially expressed genes in cells infected with avian coronavirus infectious bronchitis virus (IBV). Most members of the AP-1 transcription factors were subsequently found to be upregulated during the course of IBV and porcine epidemic diarrhea virus (PEDV) infection of cultured cells as well as in IBV-infected chicken embryos. Further characterization of the induction kinetics and functional roles of cFOS in IBV replication demonstrated that upregulation of cFOS at early to intermediate phases of IBV replication cycles suppresses IBV-induced apoptosis and promotes viral replication. Blockage of nuclear translocation of cFOS by peptide inhibitor NLSP suppressed IBV replication and apoptosis, ruling out the involvement of the cytoplasmic functions of cFOS in the replication of IBV. Furthermore, knockdown of ERK1/2 and inhibition of JNK and p38 kinase activities reduced cFOS upregulation and IBV replication. This study reveals an important function of cFOS in the regulation of coronavirus-induced apoptosis, facilitating viral replication. IMPORTANCE The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by a newly emerged zoonotic coronavirus (SARS-CoV-2), highlights the importance of coronaviruses as human and animal pathogens and our knowledge gaps in understanding the cellular mechanisms, especially mechanisms shared among human and animal coronaviruses, exploited by coronaviruses for optimal replication and enhanced pathogenicity. This study reveals that upregulation of cFOS, along with other AP-1 transcription factors, as a cell-survival strategy is such a mechanism utilized by coronaviruses during their replication cycles. Through induction and regulation of apoptosis of the infected cells at early to intermediate phases of the replication cycles, subtle but appreciable differences in coronavirus replication efficiency were observed when the expression levels of cFOS were manipulated in the infected cells. As the AP-1 transcription factors are multi-functional, further studies of their regulatory roles in proinflammatory responses may provide new insights into the pathogenesis and virus-host interactions during coronavirus infection.

A B S T R A C TCoronavirus membrane (M) protein is the most abundant structural protein playing a critical role in virion assembly. Previous studies show that the N-terminal ectodomain of M protein is modified by glycosylation, but its precise functions are yet to be thoroughly investigated. In this study, we confirm that N-linked glycosylation occurs at two predicted sites in the M protein ectodomain of infectious bronchitis coronavirus (IBV). Dual mutations at the two sites (N3D/N6D) did not affect particle assembly, virus-like particle formation and viral replication in culture cells. However, activation of the ER stress response was significantly reduced in cells infected with rN3D/N6D, correlated with a lower level of apoptosis and reduced production of pro-inflammatory cytokines. Taken together, this study demonstrates that although not essential for replication, glycosylation in the IBV M protein ectodomain plays important roles in activating ER stress, apoptosis and proinflammatory response, and may contribute to the pathogenesis of IBV.

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Coronaviruses (CoVs) are enveloped (+) ssRNA viruses of veterinary and medical importance. Because recombinant CoVs with reporter proteins fused with viral proteins are usually non-viable or unstable, a small and quantifiable epitope tag would be beneficial to CoV research. In this study, we integrated the NanoLuc Binary Technology to the reverse genetics of infectious bronchitis virus (IBV), a prototypic gammacoronavirus. The 11-amino-acid HiBiT tag was inserted to the spike (S) or membrane (M) protein, and the recombinant IBVs (rS-HiBiT and rM-HiBiT) were characterized. Compared with the rIBV-p65 control, rS-HiBiT exhibited comparable growth kinetics, whereas rM-HiBiT replicated slightly slower. The levels of HiBiT-tagged S and M proteins in the infected cells or the culture supernatant could be both rapidly (~15 min) and efficiently (30 μL sample volume) determined using the HiBiT luminescence assay. Notably, replication of the HiBiT-tagged IBV could be monitored continuously in an infected chicken embryo, and rS-HiBiT was genetically stable for at least 20 passages. By integrating the HiBiT tagging system with CoV reverse genetics, this new reporter system may facilitate future study of CoV replication and pathogenesis.