Fig. 4
Effects of m6A writer proteins on RSV gene expression. a Overexpression of m6A writer proteins enhances RSV gene expression. HeLa cells were transfected with plasmids encoding METTLE3 and/or METTL14, followed by rgRSV infection at an MOI of 0.5. At 18 h post-infection, cell lysates were harvested for western blot analysis. b Overexpression of m6A writer proteins enhances RSV expression of GFP. The GFP expression was monitored by fluorescence microscopy. Representative micrographs with ×10 magnification (scale bar of 100 μm) at 18 h post-infection were shown. c Quantification of GFP-positive cells by flow cytometry at 18 h post-infection. Fold of GFP signal compared to the control is shown. The P value (Student’s t-test) for METTL3, METTL14, and METTL3 & METTL14 is ***P = 0.000276, ***P = 0.000873, and **P = 0.00228, respectively. d Knockdown of m6A writer proteins diminishes RSV gene expression. HeLa cells were transfected with siRNA targeting METTL3 and/or METTL14, followed by rgRSV infection at an MOI of 0.5. At 18 h post-infection, cell lysates were harvested for western blot analysis. e Knockdown of m6A writer proteins diminishes GFP expression. Micrographs with ×10 magnification (scale bar of 100 μm) are shown. f Quantification of GFP-positive cells by flow cytometry. Fold of GFP signal compared to the control is shown. Flow cytometry data are expressed as mean ± standard deviation. The P value (Student’s t-test) for METTL3, METTL14, and METTL3 & METTL14 is **P = 0.00441, **P = 0.00458, and ****P = 0.000134, respectively. g Overexpression of m6A writer proteins enhances m6A level in viral RNA. Confluent A549 cells were transfected with plasmids encoding Mettl3 and Mettl14 or vector pCAGGS, followed by rgRSV infection. RSV particles were harvested and purified. The m6A content in viral RNA was determined as described in Materials and Methods. The P value (Student’s t-test) is **P = 0.00684. h Fold increase in m6A content in viral RNA compared to controls. The P value (Student’s t-test) is ***P = 0.000764. i Knockdown of m6A writer proteins reduces m6A level in viral RNA. A549 cells were transfected with siRNA targeting METTL3 and METTL14 or control siRNA, followed by rgRSV infection. RSV particles were harvested and purified. The m6A content in viral RNA was determined. The P value (Student’s t-test) is ****P = 1.29 × 10−7. j Fold reduction in m6A content in viral RNA compared to controls. The P value (Student’s t-test) is ****P = 2.45 × 10−12. Results are from three or four independent experiments
