Role of the M2 protein in influenza virus membrane fusion: effects of amantadine and monensin on fusion kinetics.

We have investigated the effects of the anti-influenza drug amantadine (AMT) and the proton-ionophore monensin on the membrane fusion activity of influenza virus in a liposomal model system, using a kinetic fluorescence lipid mixing assay. Fusion of influenza virus A/turkey/Oregon/71 (H7N3) with liposomes was slowed down in the presence of 2 microM AMT. The effect of AMT was not observed with an AMT-resistant mutant virus. Fusion inhibition by AMT was reversed by the proton-ionophore monensin. In fact, 1 microM monensin stimulated fusion of AMT-sensitive or -resistant virus, irrespective of the presence of AMT. The effects of AMT and monensin increased with increasing temperature. They were not observed at 25 degrees, but were very prominent at 45 degrees. Monensin did not influence the fusion rates of reconstituted viral envelopes (virosomes), which lack the nucleocapsid and the M1 protein. These results suggest that intraviral low pH facilitates influenza virus fusion, possibly by weakening interactions of the C-terminus of the viral hemagglutinin with the M1 protein and/or the viral nucleocapsid. The effect of AMT on the fusion capacity of influenza virus may contribute to the anti-influenza action of the drug in the early stages of cellular infection. However, the limited extent of the fusion inhibition suggests that the fusion step is unlikely to be the primary target of AMT.