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We established a reverse genetics system for the M gene of influenza A virus, using amantadine resistance as a selection criterion. Transfection of an artificial M ribonucleoprotein complex of A/Puerto Rico/8/34 (H1N1), a naturally occurring amantadine-resistant virus, and superinfection with amantadine-sensitive A/equine/Miami/1/63 (H3N8), followed by cultivation in the presence of the drug, led to the generation of a transfectant virus with the A/Puerto Rico/8/34 (H1N1) M gene. With this system, we attempted to generate a virus containing a deletion in an M-gene product (M2 protein). Viruses lacking the carboxyl-terminal Glu of M2, but not those lacking 5 or 10 carboxyl-terminal residues, were rescued in the presence of amantadine. These findings indicate that carboxyl-terminal residues of the M2 protein play an important role in influenza virus replication. The M-gene-based reverse genetics system will allow the study of different M-gene mutations to achieve a balance between attenuation and virus replication, thus facilitating the production of live vaccine strains.

Reverse genetics system for generation of an influenza A virus mutant containing a deletion of the carboxyl-terminal residue of M2 protein