Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus
Fig 5
Activation of type I and III IFN response in peroxisome-deficient human cells.
(A) Deficiency of peroxisome formation of the human fibroblast cell line ΔPex19 that does not contain peroxisomes was rescued by stable expression of Pex19 (ΔPex19+Pex19) as determined by immunofluorescence for Pex19 (red) and PMP70 (peroxisome marker, green). Nuclei were counterstained with DAPI. Scale bar, 40 μm. (B-D) Both cell lines were infected with SeV (MOI = 3) and samples were collected at indicated times points. (B) Total protein lysates of infected cells were analyzed for MxA abundance by Western blot. MxA-specific signals were quantified by using the Lab Image ID software package and values were normalized to β-actin that served as loading control. Values correspond to the mean of four independent experiments and standard errors. (C) Cell culture supernatants were harvested at given time points after SeV infection and amounts of IFN-λ1–3 were measured by ELISA. The dashed line indicates the limit of detection (LOD). (D) Fibroblasts were transfected with an IFN-λ-firefly luciferase reporter construct and 24 hours later infected with SeV. Cells were harvested at time points specified in the bottom and firefly luciferase activity was measured. Values were normalized to transfection efficiency as determined with a co-transfected SV40-based Renilla luciferase plasmid and are expressed relative to uninfected control cells (set to 1). (E) Total RNA was prepared from SeV-infected cells at time points specified in the bottom of each panel and amounts of mRNAs given on the top of each panel were determined by qRT-PCR. All data were normalized to GAPDH and uninfected control cells (set to 1). Mean values of a representative experiment conducted with technical triplicates are shown. Bars indicate the standard error. All experiments were performed at least three times.