Fig. 7
m6A-abrogating RSV mutants have defects in replication in immortalized cells. a Immunoblot analysis of RSV proteins. Confluent A549 cells were infected with each rgRSV at an MOI of 0.1, cell lysates were harvested at 18, 24, and 48 h post-infection, and RSV proteins were detected by specific antibodies against F and G protein. b GFP expression of m6A-deficient rgRSV mutants. Micrographs with ×10 magnification (scale bar of 100 μm) are shown. c Quantification of GFP-positive cells by flow cytometry. The P value (Student’s t-test) for rgRSV-G1, G2, G3, G12, and G123 at 18 h post-inoculation is ****P = 5.87 × 10−6, ****P = 7.13 × 10−5, ****P = 1.65 × 10−5, ****P = 6.49 × 10−5, and ****P = 6.98 × 10−6, respectively; at 24 h post-inoculation is **P = 0.00261, *P = 0.0418, **P = 0.00766, *P = 0.0138, and *P = 0.0230 respectively; at 48 h post-inoculation is ****P = 3.55 × 10−6, ****P = 1.82 × 10−4, ****P = 9.83 × 10–6, ****P = 7.78 × 10−5, and ****P = 2.48 × 10−5, respectively. d Single-step growth curve of m6A-deficient rgRSV mutants in A549 cells. Confluent A549 cells were infected with each rgRSV at an MOI of 1.0, supernatants were harvested, and viral titer was determined by TCID50 assay. e RSV genomic RNA replication. At 18, 24, and 48 h post-infection, total RNA was purified from rgRSV-infected cells using TRizol, and genomic RNA was quantified by real-time RT-PCR using specific primers annealing to the RSV leader sequence and GFP gene. The P value (Student’s t-test) for rgRSV-G1 at 18, 24, and 48 h is ***P = 0.000653, ****P = 8.66 × 10−5, and ****P = 1.33 × 10−5 respectively. The P value for rgRSV-G12 at 48 h is ***P = 0.000141. f RSV NS1 mRNA transcription. Viral mRNA was separated from total RNA and quantified by real-time PCR using primers annealing to the NS1. The P value (Student’s t-test) for rgRSV-G1 at 18, 24, and 48 h is ****P = 1.80 × 10−5, ****P = 1.11 × 10−4, and ***P = 0.001, respectively. g RSV G mRNA transcription. The P value (Student’s t-test) for rgRSV-G1 at 18, 24, and 48 h is ****P = 7.13 × 10−5, ****P = 5.10 × 10−6, and ****P = 1.22 × 10−6, respectively. The P value (Student’s t-test) for rgRSV-G12 at 18, 24, and 48 h is **P = 0.00942, ***P = 0.000199, and ****P = 1.35 × 10−5, respectively. Results are from three independent experiments. Flow cytometry data are expressed as mean ± standard deviation. RNA copy and viral titer are the geometric mean titer (GMT) of three independent experiments ± standard deviation. Western blots shown are the representatives of three independent experiments
