The IFIT locus is required for anti-CVB3 innate immune response in cardiomyocytes in vitro.
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(A-C) Generation of IFIT locus-edited (sgIFITs) HL-1 cells, and characterization of uninfected cells. (A) Black arrows = IFIT family genes. The locations of the PCR three primers (P1, P2 and Reverse) and sgRNA target sites spanning the IFIT locus are shown. (B) PCR results for the wt and bulk gene edited HL-1 cells are shown. (C) Western blots of the WT and IFIT locus-edited HL-1 cells. Some cells were treated for 16 hours with recombinant IFNβ (100 U/ml) to induce IFIT family proteins. GAPDH was detected as internal controls. (D-F) Phenotypical analysis of infected HL-1 cells. WT or IFITs-edited (sgIFITs) HL-1 cells were pre-treated with or without recombinant IFNβ (100 U/ml). 24 hours later, cells were infected with CVB3 at an MOI of 1. 72 hours p.i., cells and the supernatants were collected and used for subsequent studies. (D) Infectious virus titers in the supernatant were determined by plaque assays and represented as PFU/ml. Data are combined from three independent experiments (n = 3–10, geometric means). Each symbol represents an individual value. (E) Virus RNA accumulations in the cells at 72 hours p.i. were determined by real-time RT-PCR analysis. Each value was normalized to the values of Gapdh. Data are combined from two independent experiments (n = 6, geometric means). Each symbol represents an individual value. (F) Western blots of virus proteins (VP1, 3Dpol and 3A). GAPDH was detected as internal controls. (G-I) Virus titers in primary cells. Primary cardiomyocytes (G), peritoneal macrophages (H) and primary cardiac fibroblasts (I) were isolated from B6 and IFITKO mice and infected with CVB3 at MOI of 1. Then, some cells were treated with recombinant IFNβ (100 U/ml). 72 hours p.i., virus titers in the supernatants were determined by plaque assays. Data are combined from two independent experiments (n = 6–8, geometric means). Each symbol represents an individual value.