Transmembrane Domain Length of Influenza a Virus M2 Does Not Determine Its Non-Lipid Raft Localization

Influenza A virus expresses three envelope proteins, hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2). Of these, HA and NA associate with lipid rafts, whereas M2 remains in the peri-raft region. One reason for the lipid raft association of HA and NA is that they possess longer transmembrane domains (TMDs) (27 and 29 amino acids, respectively) than that of M2 (19 amino acids). Moreover, M2 localizes in the peri-raft region, despite the presence of some lipid raft-targeting features. Therefore, we introduced amino acid insertions into the N-terminal region of M2 to increase the TMD length to 22, 25, and 27 residues, and evaluated these M2-TMD mutants for their association with lipid rafts and impact on virus replication. Confocal microscopy, immunoprecipitation, and cell cytotoxicity assays showed that the cell surface expression and cytotoxic potential of M2-TMD mutants were comparable to those of wildtype M2. Based on the Triton X-100 solubility assay and colocalization analysis between lipid rafts and M2-TMD mutants, we found that the mutant proteins largely remained localized in non-raft domains. Importantly, an increase in M2-TMD length negatively influenced viral replication. These findings suggest that M2-TMD length is optimized for its proper function and does not determine its association with lipid raft domains.