Figure 1. Myotis VP35s Are Less Potent Suppressors of IFN-β Production than Extant Filoviral VP35s.

(A) IFN-β luciferase reporter assay in HEK293T cells in the presence of FLAG-tagged Myotis VP35 constructs (500 ng). Error bars represent the SEM for triplicate experiments. The uninfected empty vector control is indicated by the black bar labeled E; the remaining sampleswere infected with SeV. VP35 expression was assessed by western blot for the FLAG epitope tag. Western blot lanes align with the corresponding samples in the graph. Statistical significance was assessed using a oneway ANOVA and Tukey’s test, comparing columns to the SeV-infected control (white bar): ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. E refersto empty vector. The phylogenetic tree indicates the relationship of the tested Myotis VP35s as indicated. Blue indicates a 9 base pair (bp) deletion, black indicates no deletion, and red indicates a 39 bp deletion. Numbers on the tree graph indicate branch support as estimated from approximate likelihood ratio tests and ultrafast bootstrapping. The tree scale bar represents substitutions per site for the vertical branch lengths. (B) IFN-03b2 luciferase reporter assay in HEK293T cells in the presence of the indicated FLAG-tagged VP35 constructs at decreasing concentrations (500, 50, 5, and 0.5 ng). IFN-β promoter activity and VP35 expression were assessed as in (A). (C) Western blot analysis of IRF-3 phosphorylation in HEK293T cells transfected with decreasing concentrations of the indicated FLAG-tagged VP35 and MARV NP (mNP) constructs (2, 1, and 0.5 μg) and IRF-3 (100 ng). Western blots were performed for total IRF-3 and phospho-IRF3. The phospho-IRF3 assay was repeated twice. (D) IFN-β luciferase reporter assay in M. myotis Nep cells in the presence of FLAG-tagged Myotis VP35 constructs (500 ng). IFN-β promoter activity and VP35 expression were assessed as in (A). IFN-β promoter luciferase assays were repeated at least three times. See also Figure S1.