Fig. 7

Reduced M2 protein due to 55T mutation leads to attenuated H1N1 virus in vitro. A549 cells were infected at a MOI of 1 or 0.001 with 55T- or 55C-type RG viruses. a, d The virus titer was analyzed by quantifying the number of plaques formed at the indicated time points (hpi, hours post-infection). b, e Protein expression was determined by western blotting using specific antibodies. Total RNA was collected after 5 (c) or 36 (f) h of infection. 1F and 1R primers were used to detect different M transcripts, and 3F and 1R primers were used to determine the expression of all M transcripts. Statistical analysis was performed using paired t-tests. g WEBLOGO plots of M2 amino acid sequences based on 242 human H1N1 and 222 human H3N2 isolates. The height of each amino acid represents the corresponding frequency. Protein domains of the M2 protein are depicted. The red line shows the domain region related to the above indicated functions. a H1N1 group, t = 5.001, df = 3 and H3N2 group, t = 0.4146, df = 3. d H1N1 group, t = 2.501, df = 4 and H3N2 group, t = 1.361, df = 4. (t = t-value, df = degrees of freedom). The data represent the mean ± standard deviation (error bars) of three independent biological replicates. NS, not significant; *P < 0.05; and **P < 0.01