INFLUENZA-A VIRUS M(2) ION-CHANNEL PROTEIN - A STRUCTURE-FUNCTION ANALYSIS

A structure-function analysis of the influenza A virus M(2) ion channel protein was performed. The M(2) protein of human influenza virus A/Udorn/72 and mutants containing changes on one face of the putative alpha helix of the M(2) transmembrane (TM) domain, several of which lead to amantadine resistance when found in virus, were expressed in oocytes of Xenopus laevis. The membrane currents of oocytes expressing mutant M(2) ion channels were measured at both normal and low pH, and the amantadine-resistant mutant containing the change of alanine at residue 30 to threonine was found to have a significantly attenuated low pH activation response. The specific activity of the channel current of the amantadine-resistant mutants was investigated by measuring the membrane current of individual oocytes followed by quantification of the amount of M(2) protein expressed in these single oocytes by immunoblotting analysis. The data indicate that changing residues on this face of the putative alpha helix of the M(2) TM domain alters properties of the M(2) ion channel. Some of the M(2) proteins containing changes in the TM domain were found to be modified by addition of an N-linked carbohydrate chain at an asparagine residue that is membrane proximal and which is not modified in the wild-type M(2) protein. These N-linked carbohydrate chains were further modified by addition of polylactosaminoglycan. A glycosylated M(2) mutant protein (M(2)+V, A30T) exhibited an ion channel activity with a voltage-activated, time-dependent kinetic component. Prevention of carbohydrate addition did not affect the altered channel activity. The ability of the M(2) protein to tolerate deletions in the TM domain was examined by expressing three mutants (del(29-31),del(28-31), and del(27-31)) containing deletions of three, four, and five residues in the TM domain. No ion channel activity was detected from expression of M(2) del(29-31) and del(27-31), whereas expression of M(2) del(27-31), resulted in an ion channel activity that was activated by hyperpolarization (and not low pH) and was resistant to amantadine block. Examination of the oligomeric form of M(2) del(28-31) indicated that the oligomer is different from wild-type M(2), and the data were consistent with M(2) del(28-31) forming a pentamer.