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 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 delta) 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 microM-rimantadine or N-ethyl-rimantadine. M2 delta 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.
Schroeder
C
C
Division of Virology, National Institute for Medical Research, Mill Hill, London, U.K.
Ford
C M
CM
Wharton
S A
SA
Hay
A J
AJ
D000547:Amantadine / Q000494:pharmacology; D000818:Animals; D001190:Arylsulfonates; D002460:Cell Line; D005456:Fluorescent Dyes; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000378:metabolism*; D007473:Ion Channels / Q000502:physiology*; D008081:Liposomes / Q000378:metabolism*; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010766:Phosphorylation; D011487:Protein Conformation; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012299:Rimantadine / Q000031:analogs & derivatives / Q000494:pharmacology; D018411:Spodoptera; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000086082:Viroporin Proteins
D000547:Amantadine; D001190:Arylsulfonates; D005456:Fluorescent Dyes; D007473:Ion Channels; D008081:Liposomes; D011522:Protons; D011993:Recombinant Fusion Proteins; D012299:Rimantadine; D014763:Viral Matrix Proteins; C031540:M-protein, influenza virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C005047:pyranine