FUNCTIONAL RECONSTITUTION IN LIPID VESICLES OF INFLUENZA-VIRUS M2 PROTEIN EXPRESSED BY BACULOVIRUS - EVIDENCE FOR PROTON-TRANSFER ACTIVITY

The influenza virus M2 protein was expressed from a recombinant baculo virus in Spodoptera frugiperda Sf9 cells, purified and reconstituted i nto artificial membrane vesicles. The specific inhibitor amantadine ov ercame the toxic activity of the protein and boosted the rate of M2 sy nthesis by a factor of 10, allowing yields of about 1 mg of purified M 2 protein per g of Sf9 cells. M2 protein expressed in this system was phosphorylated and palmitoylated and displayed properties similar to t he authentic virus protein. Purified wild-type M2 protein and an amant adine-resistant mutant M2 (M2 delta) with a deletion in the trans-memb rane domain (amino acids 28 to 31) were incorporated into lipid vesicl es, which were loaded with the fluorescent pH indicator pyranine. On i mposition of an ionic gradient, M2 caused a decrease in intravesicular pH, which was susceptible to inhibition by 0.1 to 1 mu M-rimantadine or N-ethyl-rimantadine. M2 delta behaved similarly but exhibited the e xpected drug resistance. These experiments indicate that isolated M2 f unctions as an ion channel and demonstrates in vitro M2-mediated proto n translocation.