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 M-2 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 M-2 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 M. ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656-5662,2001). An M-2 protein containing a deletion in the transmembrane (TM) domain (M-2-del(29-31)) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M-2-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; N-31-S-M2WSN, a mutant in which an asparagine residue at position 31 in the M-2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from 1,WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N-31-S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M-2 residue N-31 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 M-2-del(29-31) virus. We found that the M-2-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 M-2-del(29-31) virus in 4 days after cocultivation of a 100:1 ratio of M-2-del(29-31) virus to wt virus, respectively. We conclude that the M-2 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.