title: The amphipathic helix of influenza A virus M2 protein is required for filamentous bud formation and scission of filamentous and spherical particles.
abstract: Influenza virus assembles and buds at the infected-cell plasma membrane. This involves extrusion of the plasma membrane followed by scission of the bud, resulting in severing the nascent virion from its former host. The influenza virus M2 ion channel protein contains in its cytoplasmic tail a membrane-proximal amphipathic helix that facilitates the scission process and is also required for filamentous particle formation. Mutation of five conserved hydrophobic residues to alanines within the amphipathic helix (M2 five-point mutant, or 5PM) reduced scission and also filament formation, whereas single mutations had no apparent phenotype. Here, we show that any two of these five residues mutated together to alanines result in virus debilitated for growth and filament formation in a manner similar to 5PM. Growth kinetics of the M2 mutants are approximately 2 logs lower than the wild-type level, and plaque diameter was significantly reduced. When the 5PM and a representative double mutant (I51A-Y52A) were introduced into A/WSN/33 M2, a strain that produces spherical particles, similar debilitation in viral growth occurred. Electron microscopy showed that with the 5PM and the I51A-Y52A A/Udorn/72 and WSN viruses, scission failed, and emerging virus particles exhibited a "beads-on-a-string" morphology. The major spike glycoprotein hemagglutinin is localized within lipid rafts in virus-infected cells, whereas M2 is associated at the periphery of rafts. Mutant M2s were more widely dispersed, and their abundance at the raft periphery was reduced, suggesting that the M2 amphipathic helix is required for proper localization in the host membrane and that this has implications for budding and scission.
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA.
authors.forename: Kari L
authors.initials: KL
authors.lastname: Roberts
authors.forename: George P
authors.initials: GP
authors.lastname: Leser
authors.forename: Chunlong
authors.initials: C
authors.lastname: Ma
authors.forename: Robert A
authors.initials: RA
authors.lastname: Lamb
mesh_terms: D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D009980:Influenza A virus / Q000254:growth & development / Q000502:physiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D010948:Viral Plaque Assay; D019065:Virus Assembly; D057074:Virus Release; D000086082:Viroporin Proteins
chemical_list: D050505:Mutant Proteins; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins