Figure 1.
Verification of IFN inhibitors ability to block IFN induction or IFN signaling.
(a) Small molecules reported to inhibit the IKK2 (TPCA-1) and TBK1 (BX795, MRT6884, MRT6707) components of the IFN induction pathway were tested using the A549/pr(IFN-β).GFP reporter cell-line which contains an eGFP gene under control of the IFN-β promoter. The IFN induction pathway and hence GFP expression was activated by infection with a DI rich stock of PIV5VΔC. Effect of inhibitors at various concentrations was measured by monitoring fluorescence at 18 hours post-infection. (b) Small molecules reported to inhibit the JAK1 (Cyt387, AZD1480, Ruxolitinib, Tofacitinib) component of the IFN signaling pathway were tested using the A549/pr(ISRE).GFP reporter cell-line which contains an eGFP gene under control of an ISRE promoter. The IFN signaling pathway and hence GFP expression was activated by supplementing the cell supernatant with purified IFN. Effect of inhibitors at various concentrations was measured by monitoring fluorescence at 48 hours post IFN stimulation.
Figure 2.
Effect of a panel of IFN inhibitors on BUNΔNSs virus growth in A549 cells.
The inhibitor panel consists of small molecules that target the IKK2 (TPCA-1) and TBK1 (BX795, MRT6884, MRT6707) components of the IFN induction pathway and JAK1 (Cyt387, AZD1480, Ruxolitinib, Tofacitinib) a component of the IFN signaling pathway. Effect of the inhibitors was compared with A549 cells constitutively expressing viral IFN antagonists that block IFN production (BVDV-Npro) or IFN signaling (PIV5-V). (a) Effect of various inhibitor concentrations on plaque size formation. Plaques were visualized 2 days post-infection by crystal violet staining. (b) Virus growth monitored over time in presence of 2 µM inhibitor or the equivalent volume of DMSO and pfu/ml determined from reserved supernatants by titration in Vero cells.
Figure 3.
Effect of a combination of different IFN inhibitors on BUNΔNSs growth in A549 and Vero cells.
(a) BUNΔNSs plaque formation in A549 or Vero cells in the presence of TPCA-1 or Ruxolitinib (RUX), TPCA-1 and RUX in combination with each other or PIV5-V. Each inhibitor was used at 2 µM and compared to the equivalent volume of DMSO when the inhibitors were used in combination. Plaque size formation was assessed 42 hours post infection (p.i) in A549 cells and both 42 and 72 hours p.i. in Vero cells. Plaques were visualized by crystal violet staining. (b) BUNΔNSs virus growth monitored over time in A549 cells in the presence of 4 µM RUX, the equivalent volume of DMSO or PIV5-V and Vero cells. Supernatants were reserved and pfu/ml determined by titration in Vero cells.
Figure 4.
Effect of Ruxolitinib (RUX) on BUNΔNSs and BUN-WT (wildtype) plaque formation in cell-lines derived from different mammalian species.
(a) Human cell-lines A549 and MRC5 and their derivatives constitutively expressing the viral IFN antagonist PIV5-V and (b) cell-lines derived from mouse (BalB/C), Rabbit (RK13), Dog (MDCK) and Pig (PKIBRS2). RUX was used at 4 µM and compared with an equivalent volume of DMSO. Plaques were fixed on the day indicated and visualized by immunostaining with an anti-Bunyamwera N protein antibody.
Figure 5.
Effect of Ruxolitinib (RUX) on plaque formation of a selection of viruses.
(a) RSV WT and derivatives with deleted IFN antagonists NS1 (ΔNS1) and NS2 (ΔNS2). RSV plaques were grown in the MRC5 cell-line or derivative constitutively expressing PIV5-V, fixed on the days indicated and visualized by immunostaining with an anti-RSV F protein antibody. (b) Influenza (A/PR/8/34) WT and derivative with a deleted IFN antagonist NS1 (ΔNS1). Influenza plaques were grown in the MDCK cell-line, fixed on the days indicated and visualized by immunostaining with an anti-influenza X31 antibody. (c) MeV Edmonston (Edm) and MuV Enders (End) live-attenuated vaccine strains. MeV and MuV plaques were grown in MRC5 cell-line or derivative constitutively expressing PIV5-V fixed on the day indicated and visualized by immunostaining with an anti-MeV NP or anti-MuV NP antibodies respectively. RUX was used at 4 µM and compared with the equivalent volume of DMSO.
Figure 6.
Inhibitory activity of Ruxolitinib and TPCA-1 is stable over time in cell culture.
A549 cells cultured at 37°C in the presence of media supplemented with 4 µM Ruxolitinib, 4 µM TPCA-1 or the equivalent volume of DMSO. Cell culture medium was sampled over time and stored at −80°C prior to testing the inhibitory activity of the cell culture medium containing (a) TPCA-1 in the A549/pr(IFN-β).GFP reporter cell-line or (b) Ruxolitinib in the A549/pr(ISRE).GFP reporter cell-line. The reporter assays were conducted using the standard method, briefly the A549/pr(IFN-β).GFP cell-line was activated by PIV5VΔC infection and GFP measured 18 hours post-infection and the A549/pr(ISRE).GFP cell-line activated with purified IFN and GFP measured 48 hours post-IFN treatment.