IFN-β and ISG54 promoter reporter gene assays.

HEK293T cells (5 × 10⁴) and RO6E cells (2 × 10⁵) were cotransfected using Lipofectamine 2000 with 25 ng of an IFN-β promoter-firefly luciferase reporter plasmid or an interferon-stimulated gene 54 (ISG54) promoter-firefly luciferase reporter plasmid, 25 ng of a constitutively expressing Renilla luciferase plasmid (pRL-TK; Promega), and the indicated viral protein expression plasmids (HEK293T cells, 62.5, 6.25, and 0.625 ng for VP35 and VP40 and 25, 2.5, and 0.25 ng for VP24; RO6E cells, 250, 25, and 2.5 ng for EBOV and MARV proteins and 125, 12.5, and 1.25 ng for MLAV proteins). Twenty-four hours posttransfection, cells were mock treated, SeV infected (150 hemagglutinin activity units [HAU]), or UIFN treated (1,000 U/ml). Eighteen hours postinfection or posttreatment, cells were lysed and analyzed for luciferase activity using a Dual-Luciferase reporter assay system (Promega) per the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity. Assays were performed in triplicate; error bars indicate the standard error of the mean (SEM) for the triplicate. Viral protein expression was confirmed by Western blot analysis.