Fig. 7.

No evidence supporting novel ebolavirus VP24 inhibitory activity on upstream IFN induction pathway due to indirect IFN signaling antagonism via a putative feedback loop. Reporter assays as described in Fig. 2 were conducted to rule out a putative positive IFN signaling feedback loop that indirectly induces IFNβ and thus could confound interpretation of VP24 activity data. (A and B) Cells were induced 1dpt by a high MOI of SeV (~22.2) and harvested 4hpi to assess IRF3 activity (A) and IFNβ (B) reporter activities, respectively, for early response activation. (C) Cells transfected with IFNβ or ISG54 reporter vectors alone as indicated were induced by U-IFN in the presence or absence of a neutralizing antibody (nAb) cocktail targeting IFNα, IFNβ and IFNAR1, which was diluted in medium at final concentrations of 1000U, 2500U and 20 μg/mL, respectively. (D) Cells cotransfected with IFNβ reporter vectors, and EBOV Makona VP24 vector or EV control as indicated, were induced by a high MOI of SeV in the presence or absence of increasing concentrations of nAb cocktail up to 20 μg/mL for each antibody. Values for (C) are presented as fold change of reporter activity compared to uninduced replicates. FC = fold change of positive induction relative to uninduced negative control. fLuc/rLuc = firefly/Renilla luciferase. 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 (Feagins and Basler, 2015).