title: Phosphorylation of the M2 protein of influenza A virus is not essential for virus viability.
abstract: M2 is a minor component of the influenza A virus envelope. The cytoplasmic tail of the M2 protein is posttranslationally modified in the infected cell by palmitylation and phosphorylation. The primary site for phosphorylation of the M2 cytoplasmic tail is serine 64, which is highly conserved yet not required for the activity of the M2 ion channel. Using an exogenous incorporation assay, we have shown that incorporation of M2 into virus particles is type-specific and does not require phosphorylation of the cytoplasmic tail. In addition, phosphorylation of the cytoplasmic tail is not required for the directional transport of M2 in polarized MDCK cells. Using a reverse genetics and reassortment procedure, we generated a virus (Ra) specifically mutated in segment 7 such that the M2 cytoplasmic tail could no longer be phosphorylated. The virus was found to grow as well as wild-type virus in tissue culture and in eggs, was stable on passage in these systems, and possessed no second-site mutations in the engineered RNA segment. In vivo Ra replicated in Balb/c mice at least as well as the parent strain A/WSN/33. These studies indicate that phosphorylation of the M2 cytoplasmic tail is not required for in vitro or in vivo replication of influenza A virus.
authors.affiliation: School of Animal and Microbial Sciences, University of Reading, Whiteknights, UK.
authors.forename: J M
authors.initials: JM
authors.lastname: Thomas
authors.forename: M P
authors.initials: MP
authors.lastname: Stevens
authors.forename: N
authors.initials: N
authors.lastname: Percy
authors.forename: W S
authors.initials: WS
authors.lastname: Barclay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D002642:Chick Embryo; D004285:Dogs; D009980:Influenza A virus / Q000378:metabolism*; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D016865:Reassortant Viruses / Q000378:metabolism; D016415:Sequence Alignment; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000378:metabolism*; D000086082:Viroporin Proteins
chemical_list: D007473:Ion Channels; D014763:Viral Matrix Proteins; C031540:M-protein, influenza virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins