Porcine epidemic diarrhea coronavirus infection activates caspase-8 to enhance innate immunity by blocking CYLD-mediated deubiquitination of RIG-I

Caspase-8 is best known as an initiator Caspase that induces Apoptosis. However, recent studies suggest that Caspase-8 can modulate innate Antiviral immunity in a context-dependent manner. Porcine epidemic diarrhea virus (PEDV), a coronavirus that rapidly replicates in porcine intestinal epithelial cells, triggers Apoptosis, in part through Caspase-8 activation. In this study, we investigated the role of Caspase-8 activation in regulating Antiviral responses in IPEC-DQ cells, a porcine intestinal epithelial cell line. We found that inhibition of Caspase-8 activity using Z-Vad (a pan-caspase inhibitor) and Z-IETD (a caspase-8-specific inhibitor) reduced PEDV-induced phosphorylation of TBK1, IRF3, and the p65 subunit of NF-κB, and inhibited the nuclear translocation of p65 and IRF3. Similarly, Caspase-8 knockout inhibited PEDV-induced phosphorylation of p65, IRF3, and TBK1, as well as RIG-I expression and IRF3- and NF-κB-driven luciferase activity. Notably, inhibition of Caspase-3 with Z-DEVD had no effect on PEDV-induced TBK1 and IRF3 phosphorylation. Mechanistically, Caspase-8 cleaves and inactivates CYLD, a Deubiquitinase that removes K63-linked ubiquitin from RIG-I. Caspase-8 knockout and Z-Vad blocked PEDV-induced RIG-I K63 ubiquitination. While Z-Vad inhibited cleavage of the viral N protein and promoted PEDV replication, neither Z-IETD nor Caspase-8 knockout affected N protein cleavage or virus replication. Our results suggest that Caspase-8 activation enhances innate Antiviral immunity in PEDV-infected intestinal epithelial cells without affecting viral replication, likely due to viral manipulation of IFN signaling.

CYLD; Caspase-8; Innate immunity; PEDV.