Fig. 5.

VP24 of all ebolaviruses, except RESTV in an inducer-specific manner, strongly antagonize downstream IFN signaling. Reporter assays as described in Fig. 2 were conducted to assess pan-filovirus VP24 antagonism of downstream IFN signaling, using the same signaling reporters. (A–C) Cells cotransfected with ISRE reporter vector were induced 1dpt by U-IFN (A), by poly(I:C) (B) or by tenfold increasing amounts of SeV starting from an MOI of ~0.022 (C) and harvested 24hpi. (D) Low (~0.022) or high (~22.2) MOI of SeV were used to induce cells cotransfected with IRF1 dual activity reporter vector and harvested 24hpi. (E) Cells cotransfected with an increasing concentration of indicated filovirus VP24 vectors ranging from 20 to 240 ng/well, in a VP24-specific assay conducted across two plates, along with the ISG54 reporter were induced 1dpt by a high MOI of SeV (~22.2). (F and G) Indicated FLAG-tagged VP24- and ISG54 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 (F) or for western blot expression (G); 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 VP24 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..