Fig 1. m6A-deficient virion RNAs induce higher type I IFN responses in cell culture.
(A) Western blot showing METTL3 expression in METTL3-knockdown (KD) U2OS cells and wild type U2OS cells. (B) Relative m6A level in virion RNA from RSV grown on METTL3 KD U2OS cells and wild type (wt) U2OS cells. The content of m6A level for each virion RNA was quantified by m6A methylation kit. (C) Western blot showing METTL3 expression in METTL3-KD A549 cells and control sgRNA transduced A549 cells. (D) Relative m6A level in virion RNA from RSV grown on METTL3-KD A549 cells and control sgRNA transduced A549 cells. (E) Relative m6A level in virion RNA from rgRSV mutants and wt rgRSV. rgRSV with G gene deletion mutant (rgRSV-ΔG), rgRSV-GALL(+), rgRSV-GALL(-), rgRSV-GALL(+/-), and rgRSV grew in HEp-2 cells and purified. The content of m6A level for each virion RNA was quantified. (F-H) A549 cells were transfected with 109 RNA copies (F), 2×108 RNA copies (G) and 4×107 RNA copies (H) of virion RNA of RSV grown on METTL3-KD or WT cells, IFN-β was measured at indicated time points. (I) Comparison of IFN response virion RNA of rgRSV-GALL(+/-) and WT rgRSV. A549 cells were transfected with of 2×108 RNA copies of mutant virus and IFN-β was measured. (J and K) m6A-deficient RSV RNA increases expression of RIG-I and MDA5 and induces higher IRF3 phosphorylation. A549 cells were transfected with virion RNA of RSV grown on METTL3 KD/WT cells (J) at dose of 1.0×109, 2×108 and 4×107 RNA copies, or virion RNA of rgRSV-GALL(+/-) and parental rgRSV (K) at dose of 2×108, 5×107, and 1×106 RNA copies. At indicated times, cell lysates were analyzed by Western blotting using antibodies specific to RIG-I, MDA5, IRF3, IRF3 (phosphorylated at S386) or β-actin. Data shown are mean ± s.d. of n = 6 (B, D) or n = 3 (E-I) independent experiments. Western blots are representative of n = 3 biologically independent experiments. Statistical significance was determined by two-sided Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.