Fig. 2

YTHDF1, 2, 3 (reader) proteins promote RSV replication, gene expression, and progeny virus production. a Detection of YTHDF1, 2, 3 in HeLa cells stably overexpressing YTHDF1-3. Western blot confirmed the overexpression of YTHDF1-3 proteins in HeLa cells using anti-Flag antibody. b YTHDF1, 2, 3 enhance GFP expression in rgRSV-infected cells. HeLa cells stably overexpressing these YTHDF proteins were infected with rgRSV at an MOI of 0.1, and GFP expression was monitored at the indicated times by fluorescence microscopy. Micrographs with ×10 magnification (scale bar of 100 μm) are shown. c YTHDF1, 2, 3 increase the number of GFP-positive cells quantified by flow cytometry. The P value (Student’s t-test) for YTHDF1 at 12, 18, and 24 h is **P = 0.00297, **P = 0.00145, and **P = 0.00202, respectively; for YTHDF2 at 12, 18, and 24 h is **P = 0.00318, ***P = 0.000892, and **P = 0.00312, respectively; for YTHDF3 at 12, 18, and 24 h is **P = 0.00119, ****P = 5.19 × 10⁻⁵, and ***P = 0.000647, respectively. d YTHDF1, 2, 3 enhance RSV protein expression. Total cell extracts were harvested from rgRSV-infected HeLa cells at the indicated times and subjected to western blot using antibody against RSV N, F, or G protein. Western blots shown are the representatives of three independent experiments. RSV F (F0 + F1) (e), G (f), and N (g) proteins were quantified by Image J Software. Data are expressed as mean of three independent experiments ± standard deviation. h YTHDF1, 2, 3 increase RSV progeny virus production. The release of infectious RSV particles was monitored by a single-step growth curve. Virus titer was measured by TCID₅₀. The P value (Student’s t-test) for YTHDF1 at 12, 18, and 24 h is **P = 0.0349, **P = 0.00797, and **P = 0.00741, respectively; for YTHDF2 at 12, 18, and 24 h is **P = 0.0176, ***P = 0.000279, and ***P = 1.58 × 10⁻²⁵, respectively; for YTHDF3 at 12 and 18 h is *P = 0.0487 and **P = 0.00317, respectively. i YTHDF1, 2, 3 enhance RSV genomic RNA replication. 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 YTHDF1 at 12, 18, and 24 h is ****P = 3.68 × 10⁻⁵, ****P = 5.46 × 10⁻⁵, and ****P = 4.96 × 10⁻⁵, respectively; for YTHDF2 at 12, 18, and 24 h is ***P = 0.000237, ****P = 1.48 × 10⁻⁵, and *P = 0.0184, respectively; for YTHDF3 at 12, 18, and 24 h is ***P = 0.000165, ***P = 0.000128, and *P = 0.0475, respectively. j YTHDF1, 2, 3 enhance mRNA transcription. Viral mRNA was separated from total RNA using the Dynabeads mRNA isolation kit and quantified by real-time PCR using primers annealing to the NS1 gene. The P value (Student’s t-test) for YTHDF1 at 12 and 24 h is **P = 0.00794 and ***P = 0.000218, respectively; for YTHDF2 at 12, 18, and 24 h is ****P = 4.94 × 10⁻⁶, *P = 0.0430, and ****P = 3.46 × 10⁻⁵, respectively; for YTHDF3 at 12 and 24 h is ****P = 5.93 × 10⁻⁶ and ****P = 2.39 × 10⁻⁵, respectively. k Ratio between mRNA and genomic RNA. The ratio between NS1 mRNA and genomic RNA was calculated for each cell line. All results are from three independent experiments. Flow cytometry data are expressed as mean of three independent experiments ± standard deviation. RNA copy and viral titer are the geometric mean titer (GMT) of three independent experiments ± standard deviation. The P value (Student’s t-test) for YTHDF2 at 12 and 24 h is ***P = 0.000601 and **P = 0.00130, respectively; for YTHDF3 at 24 h is ****P = 0.000599. Data were analyzed using Student’s t-test and statistical differences were indicated as *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001