Fig. 10
The attenuated phenotype of m6A mutated rgRSVs is m6A-related. a rgRSV-G1 and -G12 were less dependent on the m6A eraser protein. A549 cells were transfected with a plasmid encoding ALKBH5. At 36 h post-transfection, cells were infected with each rgRSV at an MOI of 0.5. At 18 h post-infection, cell lysates were harvested for western blot analysis. Western blot images are the representatives of three experiments. b rgRSV-G123 expression was less dependent on m6A writer protein. A549 cells were transfected with control siRNA or siRNA targeting METTL3 and METTL14. At 36 h post-transfection, cells were infected with each rgRSV at an MOI of 0.5. At 18 h post-infection, cell lysates were harvested for western blot analysis. The density of western blot was quantified by Image J software, and the ratio of the protein bands was calculated. Images are the representatives of three experiments. c Distribution of m6A peaks on the RSV mRNAs from A549 cells infected by rgRSV and rgRSV-G123. Confluent A549 cells were infected by each m6A-mutated rgRSV at an MOI of 1.0, cell lysates were harvested at 36 h post-infection. Total RNAs were extracted from cell lysates, and were enriched for mRNA by binding to oligo dT, and subjected to m6A-seq. The distribution of m6A-immunoprecipitated (IP) reads were mapped to the RSV mRNAs (pink block). The baseline distributions for mRNAs from input sample are shown as a pink line. Data presented are the mean coverage from two independent virus-infected A549 cell samples (n = 2). Red arrow indicates the m6A enrichment in G mRNA. d Virion RNA of m6A-mutated rgRSVs is defective in binding to anti-m6A antibody. Virion RNA was extracted from highly purified RSV virions. Antigenome was quantified by real-time RT-PCR. Each amount of antigenome was bound to strip wells using a RNA high binding solution, and m6A was detected using a specific capture anti-m6A antibody and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. A standard curve was generated using known m6A methylated RNA (range from 0.02 to 1 ng of m6A) as a positive control. The m6A content was calculated from each RNA samples. Data are averages of four independent experiments. The P value (Student’s t-test) for rgRSV-G12, rgRSV-G123, and rgRSV-ΔG is ***P = 0.000325, ****P = 3.09 × 10−7, and ****P = 3.74 × 10−7, respectively
