Figure 4. The genomic RNA of Zika virus (ZIKV) activates the gasdermin E (GSDME)-dependent pyroptosis through the RIG-I-caspase-8-caspase-3 pathway.

(A–C) JEG-3 cells were seeded in six-well plates and were transfected with 1 μg ZIKV 5’ untranslated region (UTR) or 3’ UTR. At 24 hr post-transfection, cells were subjected to microscopy (A), cytotoxicity (n=3) (B), and Western blot analyses (C). Scale bar, 50 μm. (D–F) RIG-I, TLR7, or TLR8 knockdown JEG-3 cells or wild-type (WT) JEG-3 cells were infected with ZIKV at a multiplicity of infection (MOI) of 1. At 24 hr post-infection, cells were subjected to microscopy (D), cytotoxicity (n=3) (E), and immunoblot analyses (F). Scale bar, 50 μm. (G–H) ZIKV-infected JEG-3 cells were infected with ZIKV. At 24 hr post-infection, the cells were subjected to cytotoxicity (n=3) (G) and Western blot analyses (H). (I–J) JEG-3 cells and RIG-I KO JEG-3 cells were seeded in six-well plates and were transfected with 1 μg ZIKV 5’ UTR. At 24 hr post-transfection, cells were subjected to cytotoxicity (n=3) (I) and Western blot analyses (J). (K–L) JEG-3 or RIG-I KO JEG-3 cells were mock-infected or infected with ZIKV at an MOI of 1. The mRNA level (K) and concentration of TNF-α in the culture supernatant (L) (n=3) was determined by RT-qPCR and enzyme-linked immunosorbent assay (ELISA), respectively. All data are presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no significance. RIG-I, retinoic acid-inducible gene I; TLR, Toll-like receptor.

Figure 4—figure supplement 1. The structural and non-structural proteins of Zika virus (ZIKV) are not capable of inducing pyroptosis in JEG-3 cells.

(A–B) JEG-3 cells were infected with ZIKV or UV-inactivated ZIKV at a multiplicity of infection (MOI) of 1. At 24 hr post-infection, the cleavage of gasdermin E (GSDME) (A), LDH release, and cell viability (n=3) (B) were analyzed. (C–D) JEG-3 cells were seeded in six-well plates followed by transfection with 2 μg of indicated plasmids. At 24 hr post-transfection, the cleavage of GSDME (C), LDH release, and cell viability (n=3) (D) were analyzed. Unpaired t-test versus control. Asterisks indicate specific bands. All data are presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. ****, p<0.0001; ns, no significance.

Figure 4—figure supplement 2. Activation of RIG-I is sufficient to induce gasdermin E (GSDME)-dependent pyroptosis.

(A) Verification of RIG-I, TLR7, and TLR8 knockdown efficiency by immunoblotting. (B–C) JEG-3 cells were seeded in six-well plate and transfected with 7.5 μl Lipofectamine 2000 Transfection Reagent, 3 μg poly (I:C) or 3 μg plasmid encoding human RIG-IN, respectively. At 24 hr post-transfection, the lactate dehydrogenase (LDH) release, cell viability (n=3) (B), and the cleavage of GSDME (C) were analyzed. (D–E) JEG-3 cells were seeded in six-well plate and transfected with 3 μg 5’ untranslated region (UTR), followed by incubation with 5 μg IFNAR1 polyclonal antibody. At 24 hr post-transfection, the LDH release and cell viability (n=3) (D), and the cleavage of GSDME (E) were analyzed. All data are presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. ***, p<0.001; ns, no significance. RIG-I, retinoic acid-inducible gene I; TLR, Toll-like receptor.