Fig 6.

Lead candidate 09167 primes the host antiviral response. (A) Host cell species dependence of 09167 antiviral activity. Avian DF-1, canine MDCK, primate Vero and CV1, and human 293T cells were infected with IAV-WSN or MeV in the presence of 1.0 and 0.33 μM 09167 or vehicle control. Progeny virus yields were normalized to titers in vehicle controls. (B) Priming of target cells with 09167 enhances antiviral activity. The compound was added to cells at 0.25 and 1.0 μM at the specified time points before or after infection with MeV, and progeny virus titers were determined at 24 hpi. Values represent means ± SD for three experiments (**, P <0.01; ***, P < 0.001). (C) Quantitative cell content mixing assay assessing MeV glycoprotein-induced virus-to-cell fusion kinetics, carried out in the presence of 1 μM 09167, vehicle (DMSO), or 75 μM fusion inhibitor AS-48 (control). At the indicated time points, reconstitution of double GFP-Renilla split-luciferase proteins, indicating cell content mixing, was determined. Values represent averages ± SD for five replicates/time point. (D) Plasmid-based IAV and MeV minigenome reporter assays to determine viral RdRp activity in the presence of increasing 09167 concentrations. Relative luciferase reporter activities were determined after 24 hours of exposure. Values represent means ± SD for at least three experiments. (E) Treatment of 293T cells with 09167 stimulates expression of several ISGs. Cells were exposed to 1.0 μM 09167 or vehicle (DMSO) for 20 h, followed by TaqMan RT-PCR quantitation of relative expression levels of a panel of genes associated with the host innate immune system. IL28A, interleukin-28; IFNB1, IFN-β; IL3RA, interleukin-3 receptor α; IRF3, interferon regulatory factor 3; IRGM, interferon-inducible protein 1; ISG15, interferon-induced 17-kDa protein; MDA5, melanoma differentiation-associated protein 5; RIG-I, retinoic acid-inducible gene 1; IFIT1, interferon-induced protein with tetratricopeptide repeats 1. Values represent averages and SD for three independent experiments; each sample was analyzed in duplicate. Symbols: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant. (F) Total lysates of cells treated as described for panel E or exposed to 50 U IFN-β were subjected to gel fractionation and immunoblotting using specific antibodies directed against RIG-I and IFIT1. As a control, blots were decorated with anti-GAPDH antibodies. *, nonspecific crossreaction.