FIG 3.

The viral RNA of virus grown in METTL3 knockout U2OS cells is defective in m⁶A methylation. (a) Western blot showing METTL3 expression in METTL3-knockout U2OS cells and wild-type U2OS cells. (b to d) Quantification of m⁶A level in virion RNA. Shown are the relative m⁶A levels in virion RNAs from SeV (b), MeV (c), hMPV (d), and VSV (e) grown on METTL3 KO/WT U2OS cells. Each virus was purified through 30 to 50% linear sucrose gradient ultracentrifugation. Virion RNA was extracted, and the total m⁶A level of each virion RNA was quantified by m⁶A RNA methylation assay. (f) Total viral RNA from VSV-infected METTL3 KO U2OS cells is defective in binding to m⁶A antibody by MeRIP assay. An MeRIP assay was carried out to determine the binding of RNA to m⁶A antibody using the Magna MeRIPTM m⁶A kit. Anti-m⁶A antibody was first conjugated to magnetic beads. Total RNA (15 μg) was extracted from VSV-infected METTL3 KO/WT U2OS cells and incubated with m⁶A antibody-associated beads at 4°C for 2 h with rotation. The RNA-associated magnetic beads were then washed for 3 times. Total RNA was extracted from beads by TRIzol reagent and quantified by real-time RT-PCR using primers annealing to VSV antigenome and genome. Data shown are the mean ± standard deviation (SD) from n = 3 (b, e, and f), n = 6 (c), or n = 4 (d) biologically independent experiments. Statistical significance was determined by two-sided Student's t test: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.