Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Apr;69(4):2306–2312. doi: 10.1128/jvi.69.4.2306-2312.1995

Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells.

R Guinea 1, L Carrasco 1
PMCID: PMC188901  PMID: 7884876

Abstract

The role that endosomal acidification plays during influenza virus entry into MDCK cells has been analyzed by using the macrolide antibiotics bafilomycin A1 and concanamycin A as selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidification of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prevented the entry of influenza virus into cells when added during the first minutes of infection. Attachment of virion particles to the cell surface was not the target for the action of bafilomycin A1. N,N'-Dicyclohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also blocked virus entry, whereas elaiophylin, an inhibitor of the plasma-proton ATPase, had no effect. The inhibitory actions of bafilomycin A1 and concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the virus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potently blocked influenza virus infection, even in the absence of antibiotics. These results indicate that a pH gradient, rather than low pH, is necessary for efficient entry of influenza virus into cells.

Full Text

The Full Text of this article is available as a PDF (552.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bowman E. J., Siebers A., Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7972–7976. doi: 10.1073/pnas.85.21.7972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carrasco L. Entry of animal viruses and macromolecules into cells. FEBS Lett. 1994 Aug 22;350(2-3):151–154. doi: 10.1016/0014-5793(94)00780-2. [DOI] [PubMed] [Google Scholar]
  3. Carrasco L., Otero M. J., Castrillo J. L. Modification of membrane permeability by animal viruses. Pharmacol Ther. 1989;40(2):171–212. doi: 10.1016/0163-7258(89)90096-x. [DOI] [PubMed] [Google Scholar]
  4. Cassell S., Edwards J., Brown D. T. Effects of lysosomotropic weak bases on infection of BHK-21 cells by Sindbis virus. J Virol. 1984 Dec;52(3):857–864. doi: 10.1128/jvi.52.3.857-864.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dean R. T., Jessup W., Roberts C. R. Effects of exogenous amines on mammalian cells, with particular reference to membrane flow. Biochem J. 1984 Jan 1;217(1):27–40. doi: 10.1042/bj2170027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Doms R. W., Helenius A., White J. Membrane fusion activity of the influenza virus hemagglutinin. The low pH-induced conformational change. J Biol Chem. 1985 Mar 10;260(5):2973–2981. [PubMed] [Google Scholar]
  7. Doms R. W. Protein conformational changes in virus-cell fusion. Methods Enzymol. 1993;221:61–72. doi: 10.1016/0076-6879(93)21007-u. [DOI] [PubMed] [Google Scholar]
  8. Dröse S., Bindseil K. U., Bowman E. J., Siebers A., Zeeck A., Altendorf K. Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. Biochemistry. 1993 Apr 20;32(15):3902–3906. doi: 10.1021/bi00066a008. [DOI] [PubMed] [Google Scholar]
  9. El Karadaghi S., Zakomirdin J. A., Shimane C., Bucher D. J., Tverdislov V. A., Kharitonenkov I. G. Interaction of influenza virus proteins with planar bilayer lipid membranes. I. Characterization of their adsorption and incorporation into lipid bilayers. Biochim Biophys Acta. 1984 Dec 5;778(2):269–275. doi: 10.1016/0005-2736(84)90368-7. [DOI] [PubMed] [Google Scholar]
  10. Fricks C. E., Hogle J. M. Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding. J Virol. 1990 May;64(5):1934–1945. doi: 10.1128/jvi.64.5.1934-1945.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fuchs P., Gruber E., Gitelman J., Kohn A. Nature of permeability changes in membrane of HeLa cells adsorbing Sendai virus. J Cell Physiol. 1980 May;103(2):271–278. doi: 10.1002/jcp.1041030212. [DOI] [PubMed] [Google Scholar]
  12. Fuchs P., Kohn A. Changes induced in cell membranes adsorbing animal viruses, bacteriophages, and colicins. Curr Top Microbiol Immunol. 1983;102:57–99. doi: 10.1007/978-3-642-68906-2_2. [DOI] [PubMed] [Google Scholar]
  13. Gluck S. L. The vacuolar H(+)-ATPases: versatile proton pumps participating in constitutive and specialized functions of eukaryotic cells. Int Rev Cytol. 1993;137C:105–137. [PubMed] [Google Scholar]
  14. Gromeier M., Wetz K. Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment. J Virol. 1990 Aug;64(8):3590–3597. doi: 10.1128/jvi.64.8.3590-3597.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guinea R., Carrasco L. Concanamycin A blocks influenza virus entry into cells under acidic conditions. FEBS Lett. 1994 Aug 8;349(3):327–330. doi: 10.1016/0014-5793(94)00695-4. [DOI] [PubMed] [Google Scholar]
  16. Guinea R., Carrasco L. Concanamycin A: a powerful inhibitor of enveloped animal-virus entry into cells. Biochem Biophys Res Commun. 1994 Jun 30;201(3):1270–1278. doi: 10.1006/bbrc.1994.1842. [DOI] [PubMed] [Google Scholar]
  17. Helenius A., Kielian M., Wellsteed J., Mellman I., Rudnick G. Effects of monovalent cations on Semliki Forest virus entry into BHK-21 cells. J Biol Chem. 1985 May 10;260(9):5691–5697. [PubMed] [Google Scholar]
  18. Helenius A., Marsh M., White J. Inhibition of Semliki forest virus penetration by lysosomotropic weak bases. J Gen Virol. 1982 Jan;58(Pt 1):47–61. doi: 10.1099/0022-1317-58-1-47. [DOI] [PubMed] [Google Scholar]
  19. Hoekstra D., Kok J. W. Entry mechanisms of enveloped viruses. Implications for fusion of intracellular membranes. Biosci Rep. 1989 Jun;9(3):273–305. doi: 10.1007/BF01114682. [DOI] [PubMed] [Google Scholar]
  20. Hoover-Litty H., Greve J. M. Formation of rhinovirus-soluble ICAM-1 complexes and conformational changes in the virion. J Virol. 1993 Jan;67(1):390–397. doi: 10.1128/jvi.67.1.390-397.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jakeman K. J., Smith H., Sweet C. Influenza virus enhancement of membrane leakiness induced by staphylococcal alpha toxin, diphtheria toxin and streptolysin S. J Gen Virol. 1991 Jan;72(Pt 1):111–115. doi: 10.1099/0022-1317-72-1-111. [DOI] [PubMed] [Google Scholar]
  22. Lanzrein M., Käsermann N., Kempf C. Changes in membrane permeability during Semliki Forest virus induced cell fusion. Biosci Rep. 1992 Jun;12(3):221–236. doi: 10.1007/BF01121792. [DOI] [PubMed] [Google Scholar]
  23. Lentz T. L. The recognition event between virus and host cell receptor: a target for antiviral agents. J Gen Virol. 1990 Apr;71(Pt 4):751–766. doi: 10.1099/0022-1317-71-4-751. [DOI] [PubMed] [Google Scholar]
  24. Madshus I. H., Olsnes S., Sandvig K. Mechanism of entry into the cytosol of poliovirus type 1: requirement for low pH. J Cell Biol. 1984 Apr;98(4):1194–1200. doi: 10.1083/jcb.98.4.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Madshus I. H., Olsnes S., Sandvig K. Requirements for entry of poliovirus RNA into cells at low pH. EMBO J. 1984 Sep;3(9):1945–1950. doi: 10.1002/j.1460-2075.1984.tb02074.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Madshus I. H. Regulation of intracellular pH in eukaryotic cells. Biochem J. 1988 Feb 15;250(1):1–8. doi: 10.1042/bj2500001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marsh M., Helenius A. Virus entry into animal cells. Adv Virus Res. 1989;36:107–151. doi: 10.1016/S0065-3527(08)60583-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Marsh M., Wellsteed J., Kern H., Harms E., Helenius A. Monensin inhibits Semliki Forest virus penetration into culture cells. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5297–5301. doi: 10.1073/pnas.79.17.5297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matlin K. S., Reggio H., Helenius A., Simons K. Infectious entry pathway of influenza virus in a canine kidney cell line. J Cell Biol. 1981 Dec;91(3 Pt 1):601–613. doi: 10.1083/jcb.91.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Matlin K. S., Reggio H., Helenius A., Simons K. Pathway of vesicular stomatitis virus entry leading to infection. J Mol Biol. 1982 Apr 15;156(3):609–631. doi: 10.1016/0022-2836(82)90269-8. [DOI] [PubMed] [Google Scholar]
  31. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  32. Meyer W. J., Gidwitz S., Ayers V. K., Schoepp R. J., Johnston R. E. Conformational alteration of Sindbis virion glycoproteins induced by heat, reducing agents, or low pH. J Virol. 1992 Jun;66(6):3504–3513. doi: 10.1128/jvi.66.6.3504-3513.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Meyer W. J., Johnston R. E. Structural rearrangement of infecting Sindbis virions at the cell surface: mapping of newly accessible epitopes. J Virol. 1993 Sep;67(9):5117–5125. doi: 10.1128/jvi.67.9.5117-5125.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Muroi M., Takasu A., Yamasaki M., Takatsuki A. Folimycin (concanamycin A), an inhibitor of V-type H(+)-ATPase, blocks cell-surface expression of virus-envelope glycoproteins. Biochem Biophys Res Commun. 1993 Jun 30;193(3):999–1005. doi: 10.1006/bbrc.1993.1724. [DOI] [PubMed] [Google Scholar]
  35. Nelson N., Taiz L. The evolution of H+-ATPases. Trends Biochem Sci. 1989 Mar;14(3):113–116. doi: 10.1016/0968-0004(89)90134-5. [DOI] [PubMed] [Google Scholar]
  36. Okada Y. Sendai virus-induced cell fusion. Methods Enzymol. 1993;221:18–41. doi: 10.1016/0076-6879(93)21005-s. [DOI] [PubMed] [Google Scholar]
  37. Pérez L., Carrasco L. Entry of poliovirus into cells does not require a low-pH step. J Virol. 1993 Aug;67(8):4543–4548. doi: 10.1128/jvi.67.8.4543-4548.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ramalho-Santos J., Nir S., Düzgünes N., de Carvalho A. P., de Lima M. da C. A common mechanism for influenza virus fusion activity and inactivation. Biochemistry. 1993 Mar 23;32(11):2771–2779. doi: 10.1021/bi00062a006. [DOI] [PubMed] [Google Scholar]
  39. Ruigrok R. W., Hewat E. A., Wade R. H. Low pH deforms the influenza virus envelope. J Gen Virol. 1992 Apr;73(Pt 4):995–998. doi: 10.1099/0022-1317-73-4-995. [DOI] [PubMed] [Google Scholar]
  40. Sato S. B., Kawasaki K., Ohnishi S. Hemolytic activity of influenza virus hemagglutinin glycoproteins activated in mildly acidic environments. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3153–3157. doi: 10.1073/pnas.80.11.3153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schlegel R., Dickson R. B., Willingham M. C., Pastan I. H. Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2291–2295. doi: 10.1073/pnas.79.7.2291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schlegel R., Willingham M., Pastan I. Monensin blocks endocytosis of vesicular stomatitis virus. Biochem Biophys Res Commun. 1981 Oct 15;102(3):992–998. doi: 10.1016/0006-291x(81)91636-3. [DOI] [PubMed] [Google Scholar]
  43. Schneider D. L. The proton pump ATPase of lysosomes and related organelles of the vacuolar apparatus. Biochim Biophys Acta. 1987;895(1):1–10. doi: 10.1016/s0304-4173(87)80013-7. [DOI] [PubMed] [Google Scholar]
  44. Seglen P. O. Inhibitors of lysosomal function. Methods Enzymol. 1983;96:737–764. doi: 10.1016/s0076-6879(83)96063-9. [DOI] [PubMed] [Google Scholar]
  45. Spruce A. E., Iwata A., Almers W. The first milliseconds of the pore formed by a fusogenic viral envelope protein during membrane fusion. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3623–3627. doi: 10.1073/pnas.88.9.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stegmann T., Booy F. P., Wilschut J. Effects of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin. J Biol Chem. 1987 Dec 25;262(36):17744–17749. [PubMed] [Google Scholar]
  47. Stegmann T., Doms R. W., Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989;18:187–211. doi: 10.1146/annurev.bb.18.060189.001155. [DOI] [PubMed] [Google Scholar]
  48. Stegmann T., White J. M., Helenius A. Intermediates in influenza induced membrane fusion. EMBO J. 1990 Dec;9(13):4231–4241. doi: 10.1002/j.1460-2075.1990.tb07871.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Weis W., Brown J. H., Cusack S., Paulson J. C., Skehel J. J., Wiley D. C. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988 Jun 2;333(6172):426–431. doi: 10.1038/333426a0. [DOI] [PubMed] [Google Scholar]
  50. White J. M. Viral and cellular membrane fusion proteins. Annu Rev Physiol. 1990;52:675–697. doi: 10.1146/annurev.ph.52.030190.003331. [DOI] [PubMed] [Google Scholar]
  51. White J., Helenius A., Gething M. J. Haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion. Nature. 1982 Dec 16;300(5893):658–659. doi: 10.1038/300658a0. [DOI] [PubMed] [Google Scholar]
  52. White J., Kartenbeck J., Helenius A. Membrane fusion activity of influenza virus. EMBO J. 1982;1(2):217–222. doi: 10.1002/j.1460-2075.1982.tb01150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. White J., Matlin K., Helenius A. Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses. J Cell Biol. 1981 Jun;89(3):674–679. doi: 10.1083/jcb.89.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wiley D. C., Skehel J. J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem. 1987;56:365–394. doi: 10.1146/annurev.bi.56.070187.002053. [DOI] [PubMed] [Google Scholar]
  55. Yoshimura A., Kuroda K., Kawasaki K., Yamashina S., Maeda T., Ohnishi S. Infectious cell entry mechanism of influenza virus. J Virol. 1982 Jul;43(1):284–293. doi: 10.1128/jvi.43.1.284-293.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yoshimura A., Ohnishi S. Uncoating of influenza virus in endosomes. J Virol. 1984 Aug;51(2):497–504. doi: 10.1128/jvi.51.2.497-504.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Young J. D., Young G. P., Cohn Z. A., Lenard J. Interaction of enveloped viruses with planar bilayer membranes: observations on Sendai, influenza, vesicular stomatitis, and Semliki Forest viruses. Virology. 1983 Jul 15;128(1):186–194. doi: 10.1016/0042-6822(83)90329-x. [DOI] [PubMed] [Google Scholar]
  58. Yu Y. G., King D. S., Shin Y. K. Insertion of a coiled-coil peptide from influenza virus hemagglutinin into membranes. Science. 1994 Oct 14;266(5183):274–276. doi: 10.1126/science.7939662. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES