FIG 3.
NP expression regulates the production of defective viral genomes and the induction of the antiviral host response. (A) Northern blot analysis of RNA extracted from A549 cells infected with IAV-NPC or IAV-NPT at an MOI of 5. Radiolabeled probes against the conserved 5′ vRNA promoter of IAV and U6 snRNA (106 nucleotides [nt]) as an internal loading control were used. (B) RT-PCR analysis for the presence of IFN-β and α-tubulin mRNA from samples used for panel A. (C) Northern blot analysis of RNA extracted from HEK-293T cells transiently expressing a truncated 200-nt-long IAV segment 6 vRNA (containing only the 100 terminal nucleotides at the 3′ and 5′ vRNA ends) together with constant amounts of IAV RdRp and increasing amounts of IAV NP. A catalytically inactive RdRp (PB1a) was used as a negative control. Radiolabeled probes against the conserved 5′ vRNA promoter of IAV and U6 snRNA (106 nt) as an internal loading control were used. (D) RT-PCR analysis for the presence of IFN-β and α-tubulin mRNA from samples used for panel C. (E) Luciferase reporter assay for IFN expression in wild-type (WT), ΔMAVS, ΔRIG-I, or ΔMDA5 A549-Dual cells infected with IAV-NPC or IAV-NPT at an MOI of 5 for 12 h. The graph shows the mean fold change of relative light units (RLU) compared to mock-infected cells from three independent biological replicates, with error bars representing the standard deviation. Statistical significance was determined by unpaired two-sample two-tailed t test. ns, not significant (P > 0.05); **, P < 0.01; ***, P < 0.001. (F) Western blot analysis of whole-cell lysates of A549 cells infected with IAV at an MOI of 5 for 8 h in the presence of increasing concentrations of Nz (0.1, 0.2, 0.3, and 0.4 μM) or BXM (1, 10, 25, and 50 μM).