A Gammacoronavirus, Avian Infectious Bronchitis Virus, and an Alphacoronavirus, Porcine Epidemic Diarrhea Virus, Exploit a Cell Survival Strategy by Upregulating cFOS To Promote Virus Replication

Abstract

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.

Induction of the Proinflammatory Chemokine Interleukin-8 Is Regulated by Integrated Stress Response and AP-1 Family Proteins Activated during Coronavirus Infection

“…The functional efficacy of cFOS is mainly driven by de novo protein synthesis, whereas cJUN is mainly activated by phosphorylation at the N-terminal Ser63 and Ser73 residues by JNK [60]. Consistently, our recent study has shown that in IBVinduced H1299 cells, cFOS mRNA was induced by~500-fold while cJUN mRNA was induced by~40-fold [45]. In addition, the basal level of cFOS protein was not detectable in the uninfected cells, but it rose to very high levels at the peak of IBV infection [45].…”

“…Use of a specific cFOS inhibitor, such as T-5224, a rationally designed small molecule that inhibits the DNA binding activity of cFOS/cJUN, would be of help in exploring this possibility further. In fact, when H1299 cells were treated with T-5224 at 12.5 µM in a previous study, IBV replication was not affected, but IBV-induced IL-8 expression was significantly reduced compared with the solvent-treated control [ 45 ]. It suggests that the DNA binding activity of cFOS is indeed required for IBV-induced IL-8 expression.…”

“…The LZD is essential for dimerization between cFOS and other AP-1 proteins, whereas the BD is required for both DNA-binding and cytosolic functions of cFOS [ 61 ]. We have previously shown that inhibition of the nuclear translocation of cFOS using a nuclear localization signal peptide (NLSP) resulted in the reduction of IBV viral protein synthesis and IBV-induced apoptosis [ 45 ]. It would be interesting to establish cFOS-knockout cell clones and use BD and LZD mutants to further investigate how the nuclear and cytoplasmic activities of cFOS would regulate cytokine production in coronavirus-infected cells.…”

“…Significant induction and activation of AP-1 proteins have been observed in coronavirus-infected cells [ 36 , 37 ] and in cells expressing coronavirus structural [ 38 , 39 ] or accessory proteins [ 40 , 41 , 42 , 43 ]. Recently we have shown that IBV infection activates both cJUN and cFOS, and their upstream MAPK signaling pathways play vital roles in regulating apoptosis and innate immunity during IBV infection [ 37 , 44 , 45 ]. However, the functional interactions between individual AP-1 family proteins in the context of coronavirus infection are not fully elucidated.…”

Animal Coronaviruses Induced Apoptosis

“…The upstream MAPK kinases and particularly MKK7 were shown to be responsible for JNK activation which served as a pro-apoptotic protein modulating the anti-apoptotic protein Bcl2; yet the pro-apoptotic activities of JNK was mediated independent of c-Jun which probably promotes cell survival [105]. A recent study revealed that ERK1/2 kinases activate cFOS which in turn suppresses apoptosis of the infected cells at early to intermediate phases of the IBV infection cycle in favor of viral replication [106].…”