Figure 2.

SARS-CoV-2 NSP5 protease-cleaved NLRP1 at the Q333 site nucleates NLRP1 inflammasome

(A) Florescence microscopy and associated quantifications of ASC-GFP specks in A549^{NLRP1+/ASC-GFP} and A549^{NLRP1−/ASC-GFP} airway epithelial cell lines infected with SARS-CoV-2 (MOI 0.05) for 24 h in the presence or absence of proteasome inhibitor bortezomib (0.1 μM) or inhibitor of the glycine N-degron pathway MLN4924 (1 μM). Images shown are from one experiment and are representative of n = 3 independent experiments; scale barss 10 μm. For quantifications, the percentage of cells with ASC complexes was determined by determining the ratios of cells positives for ASC speckles on the total cells presents in the wells. At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM.

(B) Western blot examination of NLRP1 cleavage using an anti-NLRP1 N-terminal antibody (aa 1–323) upon coincubation of SARS-CoV-2, SARS-CoV-1, or MERS-CoV 3CL (NSP5) proteases (5 μM) with A549^NLRP1+ airway epithelial cell lysates in presence or absence of the 3CL inhibitors GC-376 (10 μM) or PF-00835231 (10 μM). NLRP1 N-terminal, NLRP1 C-terminal, NSP5, and ACTIN were immunoblotted.

(C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549^{NLRP1+/ASC-GFP} airway epithelial cell lines transduced with a doxycycline (dox)-inducible plasmid encoding NSP5 or its catalytically inactive mutant NSP5^C145A. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 μm. For quantifications, the percentage of cells with ASC complexes was determined by determining the ratios of cells positives for ASC speckles on the total cells presents in the wells. At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM.

(D) Schematic representation of the approximate NLRP1 N-terminal fragment generated by NSP5 protease cut.

(E) Western blot examinations of the ability of NSP5 to cleave various NLRP1 constructs mutated in glutamine (Q) at various sites. Immunoblots show anti-N-terminal NLRP1, ACTIN, and NSP5.

(F) Florescence microscopy and associated quantifications of ASC-GFP specks in A549^{NLRP1+/ASC-GFP} or A549^{NLRP1Q333A/ASC-GFP} airway epithelial cell lines transduced with a doxycycline (dox)-inducible plasmid encoding NSP5 or its catalytically inactive mutant NSP5^C145A. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 μm. For quantifications, the percentage of cells with ASC complexes was determined by determining the ratios of cells positives for ASC speckles on the total nuclei (Blue). At least 10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM.

(G) Cell death (LDH) evaluation in A549^NLRP1+ and A549^NLRP1− airway epithelial cell lines expressing a doxycycline (dox)-inducible plasmid encoding NSP5 in the presence or absence of the proteasome inhibitor bortezomib (0.1 μM) or inhibitor of the glycine N-degron pathway MLN4924 (1 μM).

(H) Western blot examination of NLRP1 cleavage using an anti-NLRP1 N-terminal antibody (aa 1–323) after infection of A549^NLRP1−, A549^NLRP1+ A549^NLRP1Q333A, or A549^NLRP1Q130A airway epithelial cells with SARS-CoV-2 (MOI 0.05) for 24 h in the presence/absence of the proteasome inhibitor bortezomib (0.1 μM, Bort.). NLRP1 N-terminal, NSP5, and ACTIN were immunoblotted. ^∗NS: prominent nonspecific bands, not specific.

(I) Cell death (LDH) evaluation in A549^NLRP1+ and A549^NLRP1Q333A airway epithelial cell lines infected with SARS-CoV-2 (MOI 0.05) for 24 h.

(J) Western blot examination of NLRP1 cleavage using an anti-NLRP1 N-terminal antibody (aa 1–323) after infection of NHBE^WT airway epithelial cells with SARS-CoV-2 (MOI 1) for 36 h in the presence/absence of the proteasome inhibitor bortezomib (0.1 μM) or inhibitor of the glycine N-degron pathway MLN4924 (1 μM). NLRP1 N-terminal, nucleocapsid, and ACTIN were immunoblotted.

(K) Measure of cell lysis (LDH release) in NHBE^WT and NHBE^NLRP1−/− airway epithelial cells infected with SARS-CoV-2 (MOI 1) for 36 h in the presence/absence of the proteasome inhibitor bortezomib (0.1 μM) or inhibitor of the glycine N-degron pathway MLN4924 (1 μM).

Data information: images (A, C, and F) show one experiment performed 3 times. Western blot (B, E, H, and J) images are from one experiment performed 3 times. Graphs (C, G, I, and K) show data presented as means ± SEM from n = 3 independent pooled experiments; ^∗∗∗p ≤ 0.001 for the indicated comparisons with t test.