Fig. 2.

VP35 of all ebolaviruses are the most potent IFN antagonist proteins on both induction and signaling pathways. Reporter assays, using HEK293 cells in 96-well plates containing our full panel of transfected VP constructs, were conducted as described in the Materials and Methods to assess pan-filovirus VP35 activities on upstream IFN induction or downstream IFN signaling pathways (reporters shown in shaded green boxes). (A) Tenfold increasing amounts of SeV, starting from an MOI of ~0.022, were used to induce cells cotransfected with IFNβ reporter vector, which were harvested 24hpi. (B) SeV-infected cells at a high MOI (~22.2) were harvested for IFNβ reporter assay at an early timepoint post-induction. (C-H) Cells cotransfected with indicated reporters for induction (C-E), signaling (G and H) or dual activities (for IRF1, which can transactivate both IFN and ISG genes, F) were induced by a high MOI of SeV, or by dsRNA mimic poly(I:C) for IFNβ (E), as indicated, and harvested 24hpi, except for the IRF1 reporter assay (F), which was harvested 8hpi. (I and J) Indicated untagged (EBOV Makona WT) or FLAG-tagged VP35- and IFNβ reporter-cotransfected cell lysate samples derived from the same experiment were harvested 24hpi by a high MOI of SeV from replicate wells on the same plate for reporter activity (I) or for western blot expression (J); western blots were probed as indicated using an anti-FLAG antibody, as well as anti-β-actin antibody for an internal loading control; densitometry values normalized to β-actin expression and set relative to tagged EBOV Makona VP35 are shown below each protein band. FC = fold change of positive induction relative to uninduced negative control. RLU = relative light units. *=p < 0.05; **=p < 0.005; ***=p < 0.0005; ****=p < 0.0001; asterisks above horizontal lines indicate direct comparison of activity values between two antagonists (Yarilina and Ivashkiv, 2010; Hu et al., 2008; Kröger et al., 2002).