FIG 3.

NS1 C terminus of pandemic H1N1, but not SIV, inhibits the IL-1β production. (A) A schematic representation of genome composition of WT and mutant viruses. The NS1 protein encoded by different viruses is depicted. (B) PAMs were infected with Sk02 WT or Sk02-NS1/1–99 virus at an MOI of 1. At 24 hpi, cell-free supernatants were harvested for IL-1β ELISA, and the cells were lysed for Western blotting to measure the expression of pro-IL-1β and viral proteins. ns, not significant. (C) PAMs were infected with Hf09 WT or two NS1-mutant viruses, Hf09-816 and Hf09-817, at an MOI of 1 for 24 h. IL-1β ELISA and Western blotting were done as described for panel B. *, P = 0.0201; **, P = 0.0015. (D) HEK293T cells were transfected with plasmids expressing porcine NLRP3, ASC, procaspase-1, and pro-IL-1β. At 12 hpt, the cells were infected with Hf09 WT or NS1-mutant virus at an MOI of 5. IL-1β production at 12 hpi was measured by ELISA, and the expression of p20, pro-IL-1β, and viral proteins was measured by Western blotting. ***, P < 0.001. (E) PAMs were infected with Sk02, Sk02-616, or Sk02-930 at an MOI of 1 for 24 h. IL-1β ELISA and Western blotting were done as described for panel B. *, P = 0.0104; **, P = 0.0012. (F) HEK293T cells were transfected as described for panel D and infected with Sk02 or Sk02-930 at an MOI of 5 for 12 h. IL-1β ELISA and Western blotting were done as described for panel D. **, P = 0.0056. (G) HEK293T cells were transfected with plasmids expressing porcine NLRP3 inflammasome components and pro-IL-1β along with Flag-vector or Flag-tagged WT/N-terminal amino acids of Hf09 NS1. IL-1β levels and protein expression at 18 hpt were measured. **, P = 0.002; ***, P < 0.001. (H) HEK293T cells were transfected with plasmids expressing porcine NLRP3 inflammasome components and pro-IL-1β along with Myc-vector or the Myc-tagged NS1 C terminus from Hf09 or Sk02. IL-1β levels and protein expression at 18 hpt were measured as described for panel G. ***, P < 0.001.