Identification of IFNγ-stimulated genes that inhibit EBOV infection.

<p>(A) mRNA validation of hMDM profiling results for several of the top IFNγ-stimulated genes identified in our gene arrays. RNA obtained for the microarray analysis was assessed for mRNA levels of the selected genes by qRT-PCR. Results are represented as the log2 values. (B) Identification of IFNγ-stimulated genes that inhibit EBOV GP/rVSV infection. Highly permissive HEK 293T cells stably expressing TIM -1 (H3 cells) were infected with EBOV GP/rVSV 48 hours following transfection of 2 μg of ISG-RFP lentiviral constructs. Cells gated for RFP expression were assessed for EBOV GP/rVSV infection by detection of GFP. Shown is infection of EBOV GP/rVSV in ISG expressing cells relative to infection of cells transfected with a fluc-RFP expressing lentivirus (control). (C) Novel ISGs that inhibit EBOV GP/rVSV also block EBOV infection. HeLa cells were infected with EBOV 48 hours following electroporation of 5 μg of ISG-RFP lentiviral constructs. Infection was assessed by microscopy 24 hours later and percent of cells that were GFP positive were calculated by CellProfiler image analysis software. A lentiviral construct expressing IRF1 served as a positive control in these studies. (D) IRF1 knock down increases EBOV GP/rVSV infection following IFNγ stimulation. IRF1 or scrambled (Scr) siRNA were loaded into HEK 293T derived exosomes. SiRNA loaded exosomes (2.5 μg) were delivered and IFNγ added to BALB/c IFNAR^-/- peritoneal macrophages 24 hours prior to EBOV GP/rVSV infection (MOI = 0.1). Twenty-four hours following infection, total RNA was isolated from the macrophages. Amount of IRF1 expression and infection (by detection of VSV polymerase (L)) was quantified by qRT-PCR. Results represent the means ± s.e.m. Significance was determined by Student’s t-test analysis, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.