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Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture
Makoto Takeda1, Andrew Pekosz, Kevin Shuck
1Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois 60208-3500, USA.
Abstract:
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.
Insights
The M2 ion channel protein is crucial for efficient influenza virus replication. Viruses lacking M2 ion channel activity show significantly impaired growth, indicating its essential role in single-cycle replication.
Area of Science:
- Virology
- Molecular Biology
- Antiviral Research
Background:
- Amantadine targets the M2 protein's ion channel activity, inhibiting influenza A virus replication by blocking virus uncoating and Golgi pH equilibration.
- Previous studies suggested influenza viruses can replicate without M2 ion channel activity, challenging its essential role.
Purpose of the Study:
- To investigate the necessity of M2 ion channel activity for influenza virus replication.
- To determine the role of M2 residue N31 in amantadine resistance.
- To compare the replication efficiency of viruses with and without M2 ion channel activity.
Main Methods:
- Assessed amantadine sensitivity of wild-type and mutant influenza A viruses (A/WSN/33, N31S-M2WSN, MUd/WSN, A/Udorn/72).
- Generated and analyzed a mutant virus with a deleted transmembrane domain in M2 protein (M2-del(29-31)) lacking ion channel activity.
- Compared the growth kinetics and biological fitness of M2-del(29-31) virus with wild-type virus through cocultivation experiments.
Main Results:
- Identified M2 residue N31 as the sole determinant of amantadine resistance in A/WSN/33.
- Demonstrated that M2-del(29-31) virus, lacking ion channel activity, exhibited debilitated growth comparable to amantadine-treated wild-type virus.
- Showed that wild-type virus significantly outcompeted M2-del(29-31) virus in cocultivation, highlighting a fitness disadvantage for the M2-deficient virus.
Conclusions:
- The M2 ion channel protein, conserved across influenza A strains, evolved for efficient single-cycle virus replication.
- M2 ion channel activity is essential for optimal viral fitness and replication, despite previous suggestions of dispensability.
- Understanding M2 protein function provides insights into antiviral drug mechanisms and viral evolution.