Fig. 4

The role of ZIKV NS5 in the activation of the NLRP3 inflammasome. a, b HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Casp1, and pro-IL-1β. The cells were then transfected with pFlag-Env, pFlag-Capsid, pFlag-NS2A, pFlag-NS2B, pFlag-NS4B, or pFlag-NS5 (a) or transfected with pFlag-NS5 (b). IL-1β levels in the supernatants were determined by ELISA (a, b, top). The mature IL-1β, cleaved pro-Casp-1, ZIKV NS5, and GAPDH levels in the cell lysates were determined by western blot (b, bottom). c HEK293T cells were co-transfected with pcdna3.1(+)-ASC, pHA-NLRP3, or pFlag-NS5. Cell lysates were subjected to western blots (input) or IP using an IgG or anti-HA antibody and analyzed using immunoblotting with anti-HA, anti-ASC, or anti-Flag antibodies (IP). d HEK293T cells were co-transfected with pcdna3.1(+)-ASC, Flag-NLRP3 or HA-NS5 for 24 h. Cell lysates were prepared and the pellets were subjected to ASC oligomerization assays (top) or western blots (bottom). e–g THP-1 cells were infected with CT-lentivirus or NS5-lentivirus (NS5-encoding lentivirus), which were differentiated into macrophages by stimulating with TPA and then treated with 5 mM ATP for 2 h. IL-1β levels in the supernatants were determined by ELISA (e). p17 in the supernatants and pro-IL-1β, pro-Casp-1, and GAPDH in the cell lysates (Lys) were determined by western blot (f). Cell pellets were prepared and subjected to ASC oligomerization assays (g, top). ASC in the cell lysates was determined by western blot (g, bottom). h, i THP-1 cells were infected with CT-lentivirus or NS5-lentivirus, which were differentiated into macrophages by stimulating with TPA and then treated with caspase-1 inhibitor Ac-YVAD-cmk for 1 h. IL-1β levels in the supernatants were determined by ELISA (h). p17 in the supernatants and pro-IL-1β, pro-Casp-1, and GAPDH in the cell lysates (Lys) were determined by western blot (i). The number of replicates is two. Data shown are means ± s.e.m; *P < 0.05, **P < 0.01, ***P < 0.0001; ns, no significance (one-way ANOVA with Tukey’s post-hoc test)