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. 2023 Jun 8;21(6):e3002144. doi: 10.1371/journal.pbio.3002144

Fig 1. 3CLpro from SARS-CoV-2 and other coronaviruses cleaves and activates the human CARD8 inflammasome.

Fig 1

(A) A consensus Beta-CoV 3CLpro cleavage motif (upper panel; S1 Fig and Materials and methods) was used to predict two 3CLpro cleavage sites (red triangles) within the disordered “tripwire” N-terminus of human CARD8 (lower panel, green) near the described site of HIV-1pro cleavage (black triangle). Flanking residues and p-values of prediction for each site (Q37 and Q61) are shown. (B) HEK293T cells were transfected with the indicated CARD8 construct in the presence (“+”) or absence (“−”) of indicated proteases. Active (SARS-2 3CLpro) or catalytically inactive (3CLpro mutant) protease from SARS-CoV-2 was expressed as an HA-tagged fusion construct (S2 Fig). HIV-1pro was expressed from an untagged gag-pol construct. Triangles are as described in (A). (C, D) WT or CARD8 KO HEK293T cells were transfected with (“+”) or without (“−”) indicated constructs. Inflammasome activation was monitored by immunoblotting for mature IL-1β (p17) (C) or measuring culture supernatant levels of bioactive IL-1β using IL1R-expressing reporter cells (D). (E, F) CARD8 KO HEK293T cells were cotransfected with the indicated CARD8 and protease constructs and supernatant levels of bioactive IL-1β were measured by IL1R reporter assay. 3CLpros from the following viruses were used: hCoV-229E (229E), hCoV-NL63 (NL63), hCoV-HKU1 (HKU1), SARS-CoV (SARS), MERS-CoV (MERS), and mouse hepatitis virus (MHV). (D-F) Individual values (n = 3), averages, and standard deviations shown are representative of experiments performed in triplicate. Data were analyzed using two-way ANOVA with Šidák’s post-test (D, E) or one-way ANOVA with Tukey’s post-test (F). *** = p < 0.001, **** = p < 0.0001, n.s. = not significant. Data for Fig 1A, 1D, 1E, and 1F can be found in S1 Data. Beta-CoV, betacoronavirus; HIV-1pro, HIV-1 protease; IL, interleukin; MHV, mouse hepatitis virus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type; 3CLpro, 3CL protease.