T1IFN signaling into cardiomyocytes inhibits CVB3 infection in vitro.
(A-D) Generation of Ifnar1-edited (sgIfnar1) HL-1 cells, and characterization of uninfected cells. (A) Diagram of the Ifnar1 gene locus relevant to this study. Closed box = exon. The location of the sgRNA target sequence for Ifnar1 locus is shown. (B) IFNAR1 protein expression of HL-1 cells that were transfected with control vector, or vector encoding the sgRNA, was determined by flow cytometry (blue = IFNAR antibody; red = isotype control). Representative histograms are shown, along with a graph displaying the delta MFI for each population (Mean Fluorescent Intensity with IFNAR1 antibody—MFI with isotype control antibody). (C) Fold gene induction of Ifit1, Ifit2 and Ifit3 in WT and Ifnar1-edited HL-1 cells after 16 hours of IFNβ (100 U/ml) treatment. Each value was normalized to the values of Gapdh gene and divided by the values of untreated cells in each group (n = 3, Means + SEM). Note, y axes differ among the three graphs. (D) Western blots of the WT and Ifnar1-edited HL-1 cells showing constitutive and IFNβ induced IFIT protein expression. GAPDH was detected as internal controls. (E) WT or Ifnar1-edited (sgIfnar1) HL-1 cells were incubated with or without recombinant IFNβ (100 U/ml). 16 hours later, cells were infected with CVB3 at an MOI of 1. 72 hours p.i., infectious virus titers in the supernatant were determined by plaque assays. Data are combined from two independent experiments (n = 4, geometric means). Each symbol represents an individual value.