Cleavage of cellular substrate porcine gasdermin D by porcine torovirus 3C-like protease induces pyroptosis.

Torovirus (ToV), while resembling coronavirus (CoV), belongs to a distinct family Tobaniviridae in the order Nidovirales. Porcine ToV (PToV) is widespread in pig populations across many countries, yet its potential pathogenicity in pigs remains poorly understood. The viral 3C-like protease (3CLP) plays a crucial role in processing viral polyproteins and manipulating the host antiviral immune response by targeting cellular proteins through its catalytic activity. In this study, we focused on PToV 3CLP due to its unique catalytic dyad characteristics and substrate recognition properties, which are distinct from those of CoV 3CLPs. We revealed that PToV 3CLP induces pyroptosis in porcine small intestinal IPEC-J2 cells and further demonstrated that porcine gasdermin D (pGSDMD) is a cleavage substrate for PToV 3CLP associated with this process. The catalytic residues, histidine 53 and serine 160, essential for the protease activity of PToV 3CLP, were required for the cleavage of pGSDMD at two distinct sites, glutamine 193 (Q193) and glutamine 277 (Q277). One of fragments produced by PToV 3CLP cleavage, pGSDMD1-277, mimicked the activity of the N-terminal domain of pGSDMD (pGSDMD1-279) in forming pores and ultimately triggering pyroptosis. Intriguingly, these results contrast with the inhibitory effect of CoV 3CLPs on pyroptosis, previously reported to target pGSDMD at the Q193 site. The study provides additional evidence of the distinct nature of 3CLP between ToV and CoV, which may partly explain the divergent clinical manifestations and pathogenesis observed in pigs infected by these nidoviruses.