A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon
Figure 4
Antiviral activity of ASN2 is independent of interferon production and NS1.
(A) Virus titers from A549 and VERO cells infected with influenza A/WSN/33 virus (MOI = 0.01) and treated with increasing concentrations of ASN2 (6.25–50 µM) for 48 hours. (B) Plaque-reduction analysis of VERO cells infected with the indicated viruses in the presence of either DMSO or ASN2 (50 or 25 µM). Plaques were immunostained using an NP antibody. (C) Western blot analysis of cellular extracts from A549 cells infected with influenza A/WSN/33 virus (MOI = 1) and treated with ASN2 (50 µM) for 24 hours. Specific antibodies were used for each of the indicated proteins. (D) Reporter assay analysis of A549 cells transfected with IFNβ-luciferase and pRL-TK reporters, empty plasmid (-) or decreasing concentrations of NS1 (325, 32.5, and 3.25 ng) for 24 hours prior to infection with influenza A/WSN/33 virus (MOI = 1). Cells were treated with either ASN2 (50 µM) or DMSO and luciferase activity was determined 24 hours post infection. Values were normalized to Renilla luciferase activity for each sample and are represented as fold induction over uninfected DMSO-treated sample (mock). Bars represent means of triplicate values ± standard deviation.