Fig 3. Megabat CARD8 is antagonized rather than activated by coronavirus 3CLpro.
(A) Presence (filled rectangle) or absence (empty rectangle) of predicted NLRP1 or CARD8 orthologs in the indicated mammalian species. To the left is a species phylogeny. Megabat species are indicated in red. To the right is an alignment of a predicted 3CLpro cleavage site in Rousettus aegyptiacus CARD8 (red triangle indicates site and number indicates residue position). (B) Human CARD8 or R. aegyptiacus CARD8 was cotransfected with either SARS-CoV-2 (SARS-2) 3CLpro or protease from tobacco etch virus (TEVpro). For human or R. aegyptiacus CARD8 constructs labeled “WT-TEV,” a TEVpro site was introduced into the N-terminus. The red triangles and amino acid number indicate the sites of 3CLpro cleavage in human and R. aegyptiacus CARD8. The gray triangles indicate the sites of TEV protease cleavage within each CARD8 WT-TEV. (C) Mapping of the 3CLpro site within R. aegyptiacus CARD8 was performed by transfecting the indicated point mutants with SARS-CoV-2 (SARS-2) 3CLpro or TEVpro. 3CLpro and TEVpro sites are marked by triangles as in (B). (D) CARD8 KO HEK293T cells were cotransfected with R. aegyptiacus IL-1β and CASP1, along with the indicated CARD8 and protease constructs. Presence of mature IL-1 β (p17) upon TEVpro addition indicates successful reconstitution of the R. aegyptiacus CARD8 inflammasome, whereas absence of p17 upon SARS-2 3CLpro indicates antagonism of the R. aegyptiacus CARD8 inflammasome. (C) R. aegyptiacus CARD8 inflammasome activation assays were performed as in (D) with the indicated 3CLpro constructs. IL, interleukin; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; TEV, tobacco etch virus; 3CLpro, 3CL protease.