The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Figure 3
TioV-V inhibits IFN-β promoter activation by MDA5 and IL-6 signaling, but not IFN-α/β signaling.
(A) HEK-293T cells were co-transfected with reporter plasmid pISRE-Luc (300 ng/well), pRL-CMV reference plasmid (30 ng/well), and pCI-neo-3xFLAG expression vectors encoding for 3xFLAG alone or fused to MuV-V or TioV-V (300 ng/well). After 24 h, recombinant IFN-β was added at 200 IU/ml. After an additional 24 h, relative luciferase activity was determined. (B) HEK-293T cells were co-transfected with reporter plasmid pSTAT3-Luc (300 ng/well), pRL-CMV (30 ng/well), and expression vectors encoding 3xFLAG-tagged MuV-V or TioV-V (300 ng/well). At 24 h post-transfection, recombinant IL-6 was added at 10 ng/ml. After an additional 24 h, relative luciferase activity was determined. (C) HEK-293T cells were co-transfected with IFN-β-pGL3 reporter plasmid (300 ng/well), pRL-CMV (30 ng/well), expression vectors encoding 3xFLAG-tagged MDA5 (300 ng/well) and MuV-V or TioV-V (300 ng/well). After 48 h, relative luciferase activity was determined. (D) Experiment was performed as in (A), but cells were co-transfected with expression vectors encoding 3xFLAG-tagged TioV-P or TioV-W. (E) Experiment was performed as in (A), but cells were co-transfected with pCI-neo expression vector, either empty or encoding untagged MuV-V, TioV-V, TioV-P or TioV-W. (F) Experiment was performed as in (A), but cells were stimulated with recombinant IFN-λ1 at 50 ng/ml. All experiments were performed in triplicates, and data represent means ± SD. *indicates that differences observed relative to controls (none) with IFN-β, IL-6, MDA5 or IFN-λ were statistically significant (p-value<0.01).