Influenza A virus M₂ ion channel activity is essential for efficient replication in tissue culture

The amantadine-sensitive ion channel activity of influenza A virus M₂ protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M₂ protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M₂ ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656-5662, 2001). An M₂ protein containing a deletion in the transmembrane (TM) domain (M₂-del_29-31) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M₂-del_29-31 virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N₃₁S-M₂WSN, a mutant in which an asparagine residue at position 31 in the M₂ TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N₃₁S-M₂WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M₂ residue N₃₁ is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M₂-del_29-31 virus. We found that the M₂-del_29-31 virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M₂-del_29-31 virus in 4 days after cocultivation of a 100:1 ratio of M₂-del_29-31 virus to wt virus, respectively. We conclude that the M₂ ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.