THE ACTIVE OLIGOMERIC STATE OF THE MINIMALISTIC INFLUENZA-VIRUS M-2 ION-CHANNEL IS A TETRAMER

The influenza A virus M-2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of viri ons in endosomes and also modulates the pH of the trans-Golgi network in virus-infected cells, The M-2 protein is a homo-oligomer of 97 resi dues, and analysis by chemical cross-linking and SDS/PAGE indicates M- 2 forms a tetramer, However, a higher order molecular form is sometime s observed and, thus, it is necessary to determine the active form of the molecule, This was done by studying the currents of oocytes that e xpressed mixtures of the wild-type M-2 protein (epitope tagged) and th e mutant protein M-2-V27S, which is resistant to the inhibitor amantad ine. The composition of mixed oligomers of the two proteins expressed at the plasma membrane of individual oocytes was quantified after anti body capture of the cell surface expressed molecules and it was found that the subunits mixed freely, When the ratio of wild-type to mutant protein subunits was 0.85:0.15, the amantadine sensitivity was reduced to 50% and for a ratio of 0.71:0.29 to 20%, These results are consist ent with the amantadine-resistant mutant being dominant and the oligom eric state being a tetramer.