Protease-dependent and protease-independent IFN-antagonistic functions of PLPs.

(a) Primate cells (MA-104) were transfected with plasmids expressing 1 μg of WT- or CA-PLPs. EV was applied as a control. GFP-IRF-3 was coexpressed in each sample. At 24 hpi, cells were infected with IFN-inducing, recombinant Rift Valley fever virus clone 13 (RVFV Cl 13) at an MOI of 5. Cells were fixed at 8 hpi. Immunofluorescence pictures show representative results. White arrows indicate representative cells. (b) For quantification of IRF3 translocation events at least 4 representative pictures were taken. The total number of cells positive for GFP-IRF-3 and the respective PLP were determined and the number of GFP-IRF-3 nuclear translocation was calculated. The result represents one of three independently performed experiments. (c, d, e) An IFN-β promoter activation assay was conducted in HEK-293T cells to determine the anti-IFN activities of (c) SA-PLP and (d) SR-PLP and (e) SO-PLP. Cells were transfected with plasmids expressing MDA5 (IFN stimulator), Firefly- (detection of IFN-β promoter activity) and Renilla- (detection of the general transcription level) luciferases. 50 ng/24-well of EV were transfected and samples were applied as induction controls. WT- and CA-PLPs were coexpressed in a dose-dependent manner (10, 25 and 50 ng/24-well). At 17 hpt, cells were lysed and the luciferase activity was measured. Results are presented as induction relative to EV. The graph shows results of three independently performed experiments, which were all done in triplicate. Expression of the PLPs was confirmed by Western blotting. β-Actin was applied as a loading control. The error bars in each graph indicate the standard deviations of the means. Statistical significance between infected/induced EV samples and PLP-expressing cells was determined using one-way ANOVA and Dunnett-T3 post hoc test.