1887

Journal of General Virology header logo

Volume 75, Issue 12

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

Abstract

The influenza virus M2 protein was expressed from a recombinant baculovirus in 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 μ-rimantadine or -ethyl-rimantadine. M2 behaved similarly but exhibited the expected drug resistance. These experiments indicate that isolated M2 functions as an ion channel and demonstrates M2-mediated proton translocation.

  • Received:
  • Accepted:
  • Published Online:
© The Journal of General Virology 1994
Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-75-12-3477
1994-12-01
2024-04-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/75/12/JV0750123477.html?itemId=/content/journal/jgv/10.1099/0022-1317-75-12-3477&mimeType=html&fmt=ahah

References

  1. Beaven G. H., Holiday E. R. 1952; Ultraviolet absorption spectra of proteins and amino acids. Advances in Protein Chemistry 7:319–368
     [Google Scholar]
  2. Black R. A., Rota P. A., Gorodkova N., Cramer A., Klenk H.-D., Kendal A. P. 1993; Production of the M2 protein of influenza A virus in insect cells is enhanced in the presence of amantadine. Journal of General Virology 74:1673–1677
     [Google Scholar]
  3. Bukrinskaya A. G., Vorkunova N. K., Pushkarskaya N. L. 1982; Uncoating of a rimantadine-resistant variant of influenze virus in the presence of rimantadine. Journal of General Virology 60:61–66
     [Google Scholar]
  4. Carter D. B., Thomsen D. R., Im W. B., Lennon D. J., Ngo D. M., Gale W., Im H. K., Seeburg P. H., Smith M. W. 1992; Functional expression of GABAA chloride channels and benzodiazepine binding sites in baculovirus infected insect cells. BioTechnology 10:679–681
     [Google Scholar]
  5. Ciampor F., Bayley P. M., Nermut M. V., Hirst E. M. A., Sugrue R. J., Hay A. J. 1992a; Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus haemag-glutinin to the low pH conformation occurs in an acidic trans Golgi compartment. Virology 188:14–24
     [Google Scholar]
  6. Ciampor F., Thompson C. A., Hay A. J. 1992b; Regulation of pH by the M2 protein of influenza A viruses. Virus Research 22:247–258
     [Google Scholar]
  7. Dencher N. A., Burghaus P. A., Grzesiek S. 1986; Determination of the net proton-hydroxide ion permeability across vesicular lipid bilayers and membrane proteins by optical probes. Methods in Enzymology 127:746–760
     [Google Scholar]
  8. Duff K. C., Ashley R. H. 1992; The transmembrane domain of influenza A M2 protein forms amantadine-sensitive proton channels in planar lipid bilayers. Virology 190:485–489
     [Google Scholar]
  9. Duff K. C., Cudmore A. J., Bradshaw J. P. 1993; The location of amantadine hydrochloride and free base within phospholipid multilayers: a neutron and X-ray diffraction study. Biochimica et Biophysica Acta 1145:149–156
     [Google Scholar]
  10. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. 1987; Lipofection: a highly efficient, lipid-mediated DNA-trans-fection procedure. Proceedings of the National Academy of Sciences U.S.A.: 847413–7417
     [Google Scholar]
  11. Garcia M. L., Kitada M., Eisenstein H. C., Krulwich T. A. 1984; Voltage-dependent proton fluxes in liposomes. Biochimica et Biophysica Acta 766:109–115
     [Google Scholar]
  12. Grambas S., Hay A. J. 1992; Maturation of influenza A virus haemagglutinin -estimates of the pH encountered during transport and its regulation by the M2 protein. Virology 190:11–18
     [Google Scholar]
  13. Grambas S., Bennet M. S., Hay A. J. 1992; Influence of amantadine-resistance mutations on the pH regulatory function of the M2 protein of influenza A viruses. Virology 191:541–549
     [Google Scholar]
  14. Hay A. J. 1989; The mechanism of action of amantadine and rimantadine against influenza viruses. In Concepts in Virus Pathogenesi III pp 361–367 Notkins A. L., Oldstone M. B. A. Edited by New York: Springer Verlag;
     [Google Scholar]
  15. Hay A. J., Wolstenholme A. J., Skehel J. J., Smith M. H. 1985; The molecular basis of the specific anti-influenza action of amantadine. EMBO Journal 4:3021–3024
     [Google Scholar]
  16. Holsinger L. J., Lamb R. A. 1991; Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulphide bonds. Virology 183:32–43
     [Google Scholar]
  17. Indulen M. K., Polis J. J., Kanel I. A., Ryasantseva G. M., Dsegudse D. R., Kalnina V. A., Grave I. J. 1979; Antiviral activity of a series of rimantadine derivatives. In Antiviral Activity and Mode of Action of Various Chemical Compounds (in Russian) pp 34–40 Kukaine R. A. Edited by Riga: Sinatne;
     [Google Scholar]
  18. Ito T., Gorman O. T., Kawaoka Y., Bean W. J., Webster R. G. 1991; Evolutionary analysis of the influenza A virus M gene with comparison of the Ml and M2 proteins. Journal of Virology 65:5491–5498
     [Google Scholar]
  19. Kamb A., Korenbrot J. I., Kitajewski J. 1992; Expression of ion channels in cultured cells using baculovirus. Methods in Enzymology 207:423–431
     [Google Scholar]
  20. Kato N., Eggers H. J. 1969; Inhibition of uncoating of fowl plague virus by 1-adamantanamine hydrochloride. Virology 37:632–641
     [Google Scholar]
  21. Kitts P. A., Ayres M. D., Possee R. D. 1990; Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucleic Acids Research 18:5667–5672
     [Google Scholar]
  22. Kuroda K., Hauser C., Rott R., Klenk H.-D., Doerfler W. 1986; Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. EMBO Journal 5:1359–1365
     [Google Scholar]
  23. Lamb R. A., Choppin P. W. 1981; Identification of a second protein (M2) encoded by RNA segment 7 of influenza virus. Virology 112:729–737
     [Google Scholar]
  24. Luckow V. A., Summers M. D. 1989; High level expression of nonfused foreign genes with Autographa californicanuclear poly-hedrosis virus expression vectors. Virology 170:31–39
     [Google Scholar]
  25. Martin K., Helenius A. 1991; Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (Ml) promotes export and inhibits import. Cell 67:117–130
     [Google Scholar]
  26. Ohuchi M., Cramer A., Vey M., Ohuchi R., Garten W., Klenk H.-D. 1994; Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein. Journal of Virology 68:920–926
     [Google Scholar]
  27. Pinto L. H., Holsinger L. J., Lamb R. A. 1992; Influenza virus M2 protein has ion channel activity. Cell 69:517–528
     [Google Scholar]
  28. Pressman B. C. 1976; Biological applications of ionophores. Annual Review of Biochemistry 45:501–530
     [Google Scholar]
  29. Ruigrok R. W. H., Wrigley N. G., Calder L. J., Cusack S., Wharton S. A., Brown E. B., Skehel J. J. 1986; Electron microscopy of the low pH structure of influenza virus haemagglutinin. EMBO Journal 5:41–49
     [Google Scholar]
  30. Steinhauer D. A., Wharton S. A., Skehel J. J., Wiley D. C., Hay A. J. 1991; Amantadine selection of a mutant influenza virus containing an acid-stable hemagglutinin glycoprotein: evidence for virus-specific regulation of the pH of glycoprotein transport vesicles. Proceedings of the National Academy of Sciences U.S.A: 8811525–11529
     [Google Scholar]
  31. Sugrue R. J., Hay A. J. 1991; Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology 180:617–624
     [Google Scholar]
  32. Sugrue R. J., Bahadur G., Zambon M. C., Hall-Smith M., Douglas A. R., Hay A. J. 1990a; Specific structural alteration of the influenza haemagglutinin by amantadine. EMBO Journal 9:3469–3476
     [Google Scholar]
  33. Sugrue R. J., Belshe R. B., Hay A. J. 1990b; Palmitoylation of the influenza A virus M2 protein. Virology 179:51–56
     [Google Scholar]
  34. Takeuchi K., Lamb R. A. 1994; Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport. Journal of Virology 68:911–919
     [Google Scholar]
  35. Veit M., Klenk H.-D., Kendal A., Rott R. 1991; The M2 protein of influenza A virus is acylated. Journal of General Virology 72:1461–1465
     [Google Scholar]
  36. Wang C. K., Takeuchi K., Pinto L. H., Lamb R. A. 1993; The ion channel activity of influenza A virus M2 protein: characterization of the amantadine block. Journal of Virology 67:5585–5594
     [Google Scholar]
  37. Weyer U., Possee R. D. 1991; A baculovirus dual expression vector derived from the Autographa californicanuclear polyhedrosis virus polyhedrin and plO promoters : co-expression of two influenza virus genes in insect cells. Journal of General Virology 72:2967–2974
     [Google Scholar]
  38. Wharton S. A., Hay A. J., Sugrue R. J., Skehel J. J., Weis W. I., Wiley D. C. 1990; Membrane fusion by influenza viruses and the mechanism of action of amantadine. In Use of X-ray Crystallography in the Design of Antiviral Agents pp 1–12 Laver W. G., Air G. M. Edited by New York: Academic Press;
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-75-12-3477
Loading
Functional reconstitution in lipid vesicles of influenza virus M2 protein expressed by baculovirus: evidence for proton transfer activity
J. Gen. Virol. 75, 3477 (1994); https://doi.org/10.1099/0022-1317-75-12-3477
/content/journal/jgv/10.1099/0022-1317-75-12-3477
/content/journal/jgv/10.1099/0022-1317-75-12-3477
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error