FIG 2.

Influenza virus lacking the NS1 gene failed to stimulate IL-1β secretion from J774A.1 cells stably expressing the NS1 protein. (A) HEK293T cells were infected with wild-type (WT) or recombinant influenza virus lacking the NS1 gene (ΔNS1). Cell extracts were collected at 24 h postinfection, and NS1 and M2 protein expression was analyzed by immunoblotting. (B) Flt3L-cultured bone marrow pDCs were stimulated with CpG-A DNA (10 μg/ml) or WT or ΔNS1 influenza virus (MOI of 0.5) for 24 h. Cell-free supernatants were collected at 24 h poststimulation and analyzed for IFN-α by ELISA. (C) Samples from J774A.1 cells stably expressing EGFP or NS1 were analyzed by immunoblot analysis. (D and E) LPS-primed J774A.1 cells stably expressing EGFP or NS1 were stimulated with ΔNS1 or ATP. Cell-free supernatants were collected at 24 h poststimulation and analyzed for IL-1β by ELISA. Data are expressed as the means ± standard deviations. Similar results were obtained in three separate experiments. ***, P < 0.001.