Fig. 6

The 55 nucleotide is critical for the regulation of M2 splicing. Splicing of M reporters bearing each single nucleotide variant (SNV) was assessed by RT-PCR (a) and qPCR (b) using specific primers (as shown in Fig. 1e). WT: wild-type M segment, 55T or 55C: M segment with 55 mutations, 740A or 740G: M segment with a 740 mutation, 55 + 740: M segment with double mutations. c M splicing pattern HEK293 cells infected by RG viruses (MOI = 1) for 5 h. WT: wild-type virus, 55T or 55C: an RG virus containing 55 mutations. d Relative expression of M1, M2, mRNA3, and M4 determined using qPCR. Multiple comparisons between groups were performed using one-way analysis of variance. F values = 622.5 (WSN), 200.8 (pH1N1), and 411.9 (H3N2). Degrees of freedom (DFn, DFd) = (3,4). The data represent means ± standard deviations (error bars) of three independent biological replicates. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001