Functional reconstitution in lipid vesicles of influenza virus M2 protein expressed by baculovirus: evidence for proton transfer activity

Abstract

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 (M26) 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 gM-rimantadine or N-ethylrimantadine. M26 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.

Different modes of inhibition by adamantane amine derivatives and natural polyamines of the functionally reconstituted influenza virus M2 proton channel protein.

“…We showed that proton translocation into vesicles reconstituted with wild-type M2 protein was rimantadinesensitive while a rimantadine-resistant M2 variant exhibited resistance in this system (Schroeder et al, 1994). Recent electrophysiological measurements of rimantadine-sensitive proton currents in M2-expressing murine erythroleukaemia cells (Chizhmakov et al, 1996) confirm and extend these observations.…”

“…The peptide and the sheep serum were purchased from Seramun. Protein purification was monitored by silver staining and Western blots (Schroeder et al, 1994).…”

“…Expression and purification of the M2 protein sequence identical to that of influenza A\Weybridge\27 from a recombinant baculovirus (CFM2) in Trichoplusia ni cells were essentially as described in Schroeder et al (1994) except that affinitypurified sheep IgG directed against a peptide comprising the N-terminal 24 amino acids of the protein, coupled to aminolink (Pierce) was employed for immunoaffinity purification of the M2 protein. The peptide and the sheep serum were purchased from Seramun.…”

“…The intraliposomal pH was assayed by dual wavelength recordings as described (Dencher et al, 1986 ;Schroeder et al, 1994), using a Shimadzu PC5001 fluorimeter. Incubation buffer (500 µl) was equilibrated at 12 mC and readings were taken every 10 s. M2 liposomes (5-20 µl) were introduced into the buffer between two recording time-points so that the first reading after addition of vesicles was taken after 5 s. The initial internal pH of vesicles was determined in the buffer in which they were last dialysed.…”

“…The initial inhibition determined without preincubation (Table 2) rarely exceeded 50 %. Preincubation with amantadine and its derivatives was avoided because these amphiphilic compounds partition into the membrane (Duff et al, 1993) and the vesicle lumen and cause a gradual change of internal vesicle pH (Schroeder et al, 1994). Plots representing results of individual experiments with amantadine in three different ionic gradients are shown in Fig.…”