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. 2022 May 16;82(13):2385–2400.e9. doi: 10.1016/j.molcel.2022.04.033

Figure 3.

Figure 3

NSP5 protease cleaves Gasdermin D in its pore-forming domain

(A) Cell death (LDH) evaluation in A549NLRP1+, A549NLRP1−, or A549NLRP1+/GSDMD− airway epithelial cell lines infected for 24 h with SARS-CoV-2 (MOIs 0.1, 0.01, and 0.001) or stimulated with doxycycline (dox)-induced NSP5 expression.

(B) Measure of cell lysis (LDH release) and cell viability (Cell titer Glo) in NHBEWT and NHBEGSDMD−/− airway epithelial cells infected with various SARS-CoV-2 viral strains (MOI 1) for 36 h.

(C) Western blot examination of Gasdermin D (GSDMD) processing in A549NLRP1+ cells infected with SARS-CoV-2 at MOI of 0.1 for 24 h. GSDMD was immunoblotted using an anti-C-terminal antibody (recognizes full-length and C-terminal cleaved forms of GSDMD) or with an anti-GSDMD active N-terminal fragment (30 kDa) specific antibody. NLRP1, ACTIN, and SARS-CoV-2 nucleocapsid were also evaluated.

(D) Western blot examination of GSDMD and NLRP1 cleavages upon coincubation of SARS-CoV-2 NSP3 protease or SARS-CoV-2/SARS-CoV1 3CL (NSP5) proteases (5 μM) with A549NLRP1+ or A549NLRP1− cell lysates in the presence or absence of the 3CL inhibitor PF-00835231 (10 μM). GSDMD (anti C-terminal), NLRP1 N-terminal, NSP5, and ACTIN were immunoblotted.

(E) Coomassie observation of recombinant GSDMD cleavage by various amounts of SARS-CoV-2 NSP5 protease and top-down mass-spectrometry identification of GSDMD-cleaved fragments. In blue are represented the various GSDMD fragments identified upon NSP5 coincubation. In red is the NSP5 protease detected by mass spectrometry.

(F) Western blot examination and schematic representation of GSDMD cleavage by SARS-CoV-2 3CL (NSP5) or recombinant human caspase-1 (CASP1) proteases in cell lysates from A549 expressing WT GSDMD or GSDMD193A constructs. GSDMD (anti-C-terminal), NSP5, and ACTIN were immunoblotted.

(G) Cell death (LDH) evaluation in A549 cells expressing doxycycline-inducible GSDMD fragments, including GSDMD full-length (FL), caspase-1-generated active GSDMD (1–275), or NSP5-generated 1–193 and 194–484 GSDMD fragments. Cell lysis was determined 18 h after doxycycline (dox) addition in the culture medium.

(H and I) Cell death (LDH) evaluation in A549NLRP1+/GSDMD− cells complemented or not with constructs coding for WT GSDMD or GSDMD193A. Cells were transduced with dox-inducible NSP5 plasmids, treated with the NLRP1 activator Val-boro (10 μM) or infected (I) with SARS-CoV-2 (MOI 0.01). Cell lysis was determined 18 h after doxycycline (dox) addition, 10 h after Val-boro addition in the culture medium, or 24 h after infection.

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