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Division of Virology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
The influenza virus M2 protein was expressed from a recombinant baculovirus in Spodoptera frugiperda Sf9 cells, purified and reconstituted into artificial membrane vesicles. The specific inhibitor amantadine overcame the toxic activity of the protein and boosted the rate of M2 synthesis by a factor of 10, allowing yields of about 1 mg of purified M2 protein per g of Sf9 cells. M2 protein expressed in this system was phosphorylated and palmitoylated and displayed properties similar to the authentic virus protein. Purified wild-type M2 protein and an amantadine-resistant mutant M2 (M2
) with a deletion in the trans-membrane domain (amino acids 28 to 31) were incorporated into lipid vesicles, which were loaded with the fluorescent pH indicator pyranine. On imposition of an ionic gradient, M2 caused a decrease in intravesicular pH, which was susceptible to inhibition by 0.1 to 1 µM-rimantadine or N-ethylrimantadine. M2 behaved similarly but exhibited the expected drug resistance. These experiments indicate that isolated M2 functions as an ion channel and demonstrates in vitro M2-mediated proton translocation.
Present address: Institut für Virologie, Medizinische Fakultät (Charité) der Humboldt-Universität zu Berlin, Schumannstrasse 20/21, 10098 Berlin, Germany.
Present address: Waite Agricultural Research Institute, The University of Adelaide, Glen Osmond, South Australia 5064.
Received 9 May 1994;
accepted 29 July 1994.
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