Mutations in the Membrane-Proximal Region of the Influenza A Virus M2 Protein Cytoplasmic Tail Have Modest Effects on Virus Replication

Influenza A virus encodes M2, a proton channel that has been shown to be important during virus entry and assembly. In order to systematically investigate the role of the membrane-proximal residues in the M2 cytoplasmic tail in virus replication, we utilized scanning and directed alanine mutagenesis in combination with transcomplementation assays and recombinant viruses. The membrane-proximal residues 46 to 69 tolerated numerous mutations, with little, if any, effect on virus replication, suggesting that protein structure rather than individual amino acid identity in this region may be critical for M2 protein function. Influenza A virus is a member of the Orthomyxoviridae fam-ily whose genome consists of 8 negative-sense RNA segments which encode 10 or 11 proteins. The highly conserved integral membrane protein M2 is encoded by segment 7 via a spliced mRNA, consists of 97 amino acids, and forms disulfide-linked tetramers which have a pH-gated, proton-selective ion channel activity. M2 is a type III integral membrane protein with an extracellular amino terminus and an intracellular carboxy ter-minus. M2 is required for virus entry, during which it translo