ANALYSIS OF THE POSTTRANSLATIONAL MODIFICATIONS OF THE INFLUENZA-VIRUS M(2) PROTEIN

The sites of posttranslational modifications of the influenza A virus M(2) protein were examined, and the effect of these modifications on the M(2) protein ion channel activity was analyzed. Cysteine residues 17 and 19 in the M(2) protein ectodomain form disulfide bonds. The cytoplasmic tail is posttranslationally modified by palmitoylation, and mutagenic studies support the view that cystein residue 50 is the site for fatty acylation. In addition, the cytoplasmic tail of the M(2) protein was found to be posttranslationally modified by the addition of phosphate to specific serine residues. Site-directed mutagenesis of serine residues in the M(2) protein cytoplasmic tail, combined with phosphoamino acid analysis, indicated that serine residue 64 is the predominant site for phosphorylation but that serine residues 82, 89, and 93 were also phosphorylated but to much less extents. Disulfide-bond formation, palmitoylation, and phosphorylation occurred on M(2) protein expressed in mammalian cells infected with influenza virus, in mammalian cells in which the M(2) protein was expressed from DNA expression vectors, and when the M(2) protein was expessed in oocytes of Xenopus laevis. The memebrane currents of oocytes of Xenopus laevis expressing wild-type and site-specifically altered forms of the M(2) protein, to ablate posttranslational modifications, indicated that none of the posttranslational modifications significantly affected the ion channel activity of the M(2) protein in oocytes. Therefore, these data do not indicate a functional role for posttranslational modifications of the M(2) protein in its ion channel activity.