Figure 2. NLRP1 inhibition during MVA virus infection is mediated by F1L.

(A) N/TERT1 cells were infected with WT, ΔF1L, or ΔF1L + rF1L MVA virus at MOI 5 for 4, 1, or 2 h, and then stimulated with ANS for 2 h, VbP for 7 h, and poly(I:C) for 6 h, respectively. IL-18 release was measured by ELISA. Data from three independent experiments were summarized and are presented as mean values ± SEM. Data are analyzed by using a two-way ANOVA with Šídák’s multiple comparisons test. (B) N/TERT1 cells were transfected with in vitrotranscribed mRNA encoding mScarlet or F1L for 6 h and stimulated with ANS for 2 h. IL-1β release was measured by ELISA. Data from three independent experiments were summarized and are presented as mean values ± SEM. Data are analyzed by using a two-way ANOVA with Šídák’s multiple comparisons test. (C) NLRP1^KO N/TERT1 cells complemented with NLRP1 were transfected with mRNA encoding mScarlet or F1L for 6 h and then stimulated with ANS for 2 h or transfected for 4 h and then stimulated with VbP for 4 h. IL-1β release was measured by ELISA. Data from seven independent experiments were summarized and are presented as mean values ± SEM. Data are analyzed by using a two-way ANOVA with Šídák’s multiple comparisons test. (D) NLRP1^KO N/TERT1 cells complemented with NLRP1 or NLRP3 were transfected with mRNA encoding mScarlet or F1L for 6 h and then stimulated with ANS or Nigericin for 2 h. IL-1β release was measured by ELISA. Data from six independent experiments were summarized and are presented as mean values ± SEM. Data shown in (D) include data from (C) (Control and ANS stimulation for NLRP1-expressing cells). Data are analyzed by using a two-way ANOVA with Šídák’s multiple comparisons test. (E, F) N/TERT1 cells were transfected with mRNA encoding mScarlet or F1L and its indicated variants for 6 h and then stimulated with ANS for 2 h. IL-1β release was measured by ELISA. Data from three independent experiments were summarized and are presented as mean values ± SEM. Data are analyzed by using a two-way ANOVA with Dunnett’s multiple comparisons test. (G) HEK293T cells expressing ASC-mCherry and NLRP1 or NLRP3 were transfected with plasmids encoding YFP or YFP-F1L and stimulated the next day with ANS or Nigericin for 2 h. ASC specking was analyzed by flow cytometry. The graph represents the percentage of specking cells in the YFP-positive population. Data from four independent experiments were summarized and are presented as mean values ± SEM. Data are analyzed by using a two-way ANOVA with Šídák’s multiple comparisons test. (H, I) HEK293T cells expressing ASC-mCherry and NLRP1 were transfected with the indicated constructs and treated and analyzed as in (G). Data from four (H) or three (I) independent experiments were summarized and are presented as mean values ± SEM. Data are analyzed by using a two-way ANOVA with Dunnett’s multiple comparisons test. (J) N/TERT1 cells were treated with Z-VAD-FMK for 6 h (Control), with Z-VAD-FMK immediately followed by infection with WT, ΔF1L, or ΔF1L + rF1L MVA virus at MOI 5 for 4 h and then stimulated with ANS for 2 h, or treated with Z-VAD-FMK for 4 h, followed by stimulation with ANS for 2 h. Data are representative of three independent experiments with similar results.