Interferon-? Inhibits Ebola Virus Infection

Rhein, Bethany A.; Powers, Linda S.; Rogers, Kai; Anantpadma, Manu; Singh, Brajesh K.; Sakurai, Yasuteru; Bair, Thomas; Miller-Hunt, Catherine; Sinn, Patrick; Davey, Robert A.; Monick, Martha M.; Maury, Wendy

Type I or type II IFN treatment of macrophages elicits expression of hundreds of IFN stimulated genes (ISGs) and suppresses the expression of an additional smaller group of genes. Production of ISG proteins alters macrophage function and protects cells from viral infection [35] . While gene sets activated by these two types of IFNs overlap, both type I and II IFNs also stimulate unique subsets of genes [17, 18, 36] . To determine the impact of IFNγ stimulation on gene expression in our macrophage cultures, we performed microarray analyses. We identified 268 genes with at least two-fold increased or decreased expression in IFNγ-treated six-day- Table) . In parallel, gene arrays were performed in IFNγ-treated alveolar macrophages and 45 genes were identified to have altered expression (S5 Fig and S2 Table) . Forty-one of the significantly altered genes were identified in both arrays ( Fig 3B and S6A Fig) . Through pathway analysis of the gene profiles, we discovered that the top IFNγ-upregulated genes in our arrays are involved in immune responses, development, signal transduction, ATP metabolism, or transcription (S6B Fig). Our arrays identified IFNγ-enhanced expression of ISGs known to be primary-response genes, such as the transcription factors IRF1 and IRF9, and more poorly studied secondaryresponse ISGs, such as the p65 GBPs and the apolipoprotein L family of proteins that are thought be regulated by IRFs [37] . Consistent with previous reports, a number of chemokines Interferon-γ Inhibits Ebola Virus Infection (CXCL9, CXCL10, CCL8 and CXCL11) and complement components (C1s and C1r) were also highly upregulated by IFNγ [14, 16, 17, 38] . Our array findings were validated by examining mRNA levels of several of the top ISGs, including IRF1, GBP4, GBP5, IFIT3, RARRES3, and VAMP5 by qRT-PCR ( Fig 4A and S5B Fig) . 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, *p < 0.05, **p < 0.01, ***p < 0.001. To assess the importance of some of our most significantly upregulated ISGs in controlling EBOV GP/rVSV and EBOV infection, IFNγ-upregulated genes were transfected into cells and their effect on virus infection was assessed. The panel of ISGs included both well-studied and poorly characterized IFNγ-responsive genes. Eight lentiviral constructs that expressed an ISG and red fluorescent protein (RFP) were assessed for inhibition of EBOV GP/rVSV or EBOV in highly permissive cells [23] .