SARS-CoV-2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage (2021)
Figure 3

Figure 3. SARS-CoV-2 nucleocapsid interacts with GSDMD.

(A) Plasmids encoding FLAG-tagged full-length GSDMD were co-transfected with plasmids encoding Myc-tagged SARS-CoV-1 nucleocapsid or SARS-CoV-2 nucleocapsid into HEK293T cells for 24 h, followed by immunoprecipitation with antibody against Myc or isotype IgG. Input and precipitates were immunoblotted as indicated.

(B) Plasmids encoding FLAG-tagged full-length GSDMD, GSDMD C-terminus and Myc-tagged nucleocapsid were transfected into HEK293T cells individually. Exogenous proteins were purified using FLAG or Myc targeting antibodies coupled to magnetic beads 24 h post transfection. Purified proteins were then mixed and incubated for 2h, followed by immunoprecipitation with antibody against FLAG or isotype IgG. Input and precipitates were immunoblotted as indicated.

(C) GST-GSDMD, His-tagged nucleocapsid and control GST-3C protease (GST-3Cpro) were purified from E. coli BL21, followed by a GST-pulldown assay. Input and precipitates were visualized by Coomassie blue staining.

(D) GSDMD+/+ and GSDMD-/- THP-1 cells were transfected with plasmids encoding nucleocapsid for 24 h, followed by cell fixation, permeabilization and stain with antibodies against nucleocapsid and GSDMD. The nucleus was counterstained with DAPI. Scale bar: 5 μm.

(E, F) THP-1 (E) or human CD14+ monocytes (F) were transfected with plasmids encoding FLAG-tagged nucleocapsid for 36 h, followed by pretreatment of 1 μg/ml LPS for 3 h and 10 μM nigericin stimulation for 30 min. Cells were lysed and immunoprecipitated with antibody against FLAG or isotype IgG. Input and precipitates were immunoblotted as indicated.

(G) Schemes for GSDMD (left panel) and SARS-CoV-2 nucleocapsid (right panel) truncations.

(H) Plasmids encoding DNA binding domain (BD)-tagged nucleocapsid truncations and activation domain (AD)-tagged GSDMD C-terminus truncations were co-transfected into yeast strain AH109 and double positive clones were grown on plates containing X-α-gal. The intensity of blue color was calculated and shown as scores for both dot size and color. Plasmids encoding BD-p53 and AD-large T antigen were co-transfected as a positive control.

(I) GST-GSDMD and control GST were mixed with HEK293T cell lysate containing FLAG-tagged full-length nucleocapsid or truncated nucleocapsid lacking amino acids 70-160 and 290-360, followed by a GST-pulldown assay. Input and precipitates were immunoblotted as indicated.

(J, K) THP-1 cells stably expressing full-length nucleocapsid or truncated nucleocapsid lacking amino acids 70-160 and 290-360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. An ELISA assay was performed to determine supernatant IL-1β levels (J) and a cell viability assay was conducted to verify the percentage of viable cells through checking cellular ATP levels (K). For J, K, Control vector used here was empty vector corresponded to the nucleocapsid-containing vector. For J, K, Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05. Results were repeated for three times with similar results.

Data information: Control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05. Results were repeated for three times with similar results. Experiments were repeated for three times with similar results.