Figure 4. ZIKV NS3 inhibits NLRP3-dependent caspase-1 activation and IL-1β secretion from bone marrow-derived macrophages and brain mixed glial cells. (A, B) Immunoblots from BMDMs (A) or mixed glial cells (B) infected with mock or ZIKV (MOI=10, 20 h), washed, and then transfected with poly dA:dT (1 μg/ml, 6 h). (C-E) Quantification of IL-1β in culture supernatants of BMDMs transfected with GFP control, ZIKV NS1 (C), ZIKV NS3 (D) or ZIKV NS5 (E) expression plasmid (0.5 µg) for 40 h, then treated with LPS (0.25 µg/ml, 3 h) and ATP (2 mM, 1 h) (n=5, C; n=6, D-E). (F-H) Immunoblots in culture supernatants (Sup) or RT-PCR for the mRNA expression of target genes in the cellular extracts from BMDMs treated as in (C-E). (I) Immunoblots from BMDMs transfected with GFP or ZIKV NS3 expression plasmid for 6 h, washed, incubated for additional 40 h, and then transfected with poly dA:dT (1 μg/ml, 6 h). (J) Immunoblots of the cell lysates of 293T cells transfected with NLRP3 or NS3 plasmids for 6 h, washed, and further incubated for 40 h with or without serine protease inhibitor (0.5 µM, PMSF).

Asterisks indicate significant differences (^*p<0.05; n.s., not significant).