UC San Diego

Within the evolutionary arms race between viruses and their hosts, hosts have evolved innate immune sensors to detect viral protease activity. For instance, human caspase recruitment domain-containing protein 8 (CARD8) has a disordered N-terminal region that contains mimics of the preferred cleavage site of coronavirus 3CL proteases (3CLpro). The cleavage of the N-terminal region by viral proteases results in pyroptosis and pro-inflammatory cytokine release. Interestingly, unlike human CARD8, other vertebrate CARD8 homologs have an N-terminal ubiquitin-like (Ubl) domain. However, innate immune sensing of viral proteases by Ubl-containing CARD8 (Ubl-CARD8) homologs has not been investigated. Previous studies have highlighted the ability of viral deubiquitinases (DUBs) to cleave ubiquitin and Ubl domains, raising the possibility of innate immune sensing of viral DUBs via Ubl-CARD8 homologs. Therefore, we sought to investigate the capabilities of Ubl-CARD8 homologs to sense linear proteases such as coronavirus 3CLpro and have an expanding sensing ability for viral DUBs. Our findings reveal that viral DUBs selectively cleave the C-terminal conjugation motif (5’-XLRGG | X-3’) of the Ubl domain within chicken CARD8. Furthermore, cleavage of a chimeric Ubl-CARD8, human CARD8 with the chicken N-terminal Ubl domain, by viral DUBs activated the CARD8 inflammasome. The cleavage of chicken CARD8 by viral DUBs and coronavirus 3CLpro paired with the subsequent inflammasome activation broadens viral sensing capabilities of Ubl-CARD8, which could be evolutionarily beneficial for hosts. Overall, these findings pave the way for a better understanding of how hosts can detect and respond to viruses by taking advantage of viral protease specificity.