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. 2002 Nov;83(5):2652–2666. doi: 10.1016/S0006-3495(02)75275-2

Measuring pKa of activation and pKi of inactivation for influenza hemagglutinin from kinetics of membrane fusion of virions and of HA expressing cells.

Aditya Mittal 1, Tong Shangguan 1, Joe Bentz 1
PMCID: PMC1302350  PMID: 12414698

Abstract

The data for the pH dependence of lipid mixing between influenza virus (A/PR/8/34 strain) and fluorescently labeled liposomes containing gangliosides has been analyzed using a comprehensive mass action kinetic model for hemaglutinin (HA)-mediated fusion. Quantitative results obtained about the architecture of HA-mediated membrane fusion site from this analysis are in agreement with the previously reported results from analyses of data for HA-expressing cells fusing with various target membranes. Of the eight or more HAs forming a fusogenic aggregate, only two have to undergo the "essential" conformational change needed to initiate fusion. The mass action kinetic model has been extended to allow the analysis of the pKa for HA activation and pKi for HA inactivation. Inactivation and activation of HA following protonation were investigated for various experimental systems involving different strains of HA (A/PR/8/34, X:31, A/Japan). We find that the pKa for the final protonation site on each monomer of the trimer molecule is 5.6 to 5.7, irrespective of the strain. We also find that the pKi for the PR/8 strain is 4.8 to 4.9. The inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X:31 HA-expressing cells fusing with red blood cells, were both found to be of the order of 10(-4) s(-1). This number appears to be the minimal rate for HA's essential conformational change at low HA surface density. At high HA surface densities, we find evidence for cooperativity in the conformational change, as suggested by other studies.

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Selected References

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  1. Alford D., Ellens H., Bentz J. Fusion of influenza virus with sialic acid-bearing target membranes. Biochemistry. 1994 Mar 1;33(8):1977–1987. doi: 10.1021/bi00174a002. [DOI] [PubMed] [Google Scholar]
  2. Armstrong R. T., Kushnir A. S., White J. M. The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition. J Cell Biol. 2000 Oct 16;151(2):425–437. doi: 10.1083/jcb.151.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bentz J., Ellens H., Alford D. An architecture for the fusion site of influenza hemagglutinin. FEBS Lett. 1990 Dec 10;276(1-2):1–5. doi: 10.1016/0014-5793(90)80492-2. [DOI] [PubMed] [Google Scholar]
  4. Bentz J. Intermediates and kinetics of membrane fusion. Biophys J. 1992 Aug;63(2):448–459. doi: 10.1016/S0006-3495(92)81622-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bentz J. Membrane fusion mediated by coiled coils: a hypothesis. Biophys J. 2000 Feb;78(2):886–900. doi: 10.1016/S0006-3495(00)76646-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bentz J. Minimal aggregate size and minimal fusion unit for the first fusion pore of influenza hemagglutinin-mediated membrane fusion. Biophys J. 2000 Jan;78(1):227–245. doi: 10.1016/S0006-3495(00)76587-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bentz J., Mittal A. Deployment of membrane fusion protein domains during fusion. Cell Biol Int. 2000;24(11):819–838. doi: 10.1006/cbir.2000.0632. [DOI] [PubMed] [Google Scholar]
  8. Blumenthal R. Cooperativity in viral fusion. Cell Biophys. 1988 Jan-Jun;12:1–12. doi: 10.1007/BF02918347. [DOI] [PubMed] [Google Scholar]
  9. Blumenthal R., Sarkar D. P., Durell S., Howard D. E., Morris S. J. Dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events. J Cell Biol. 1996 Oct;135(1):63–71. doi: 10.1083/jcb.135.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bullough P. A., Hughson F. M., Skehel J. J., Wiley D. C. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature. 1994 Sep 1;371(6492):37–43. doi: 10.1038/371037a0. [DOI] [PubMed] [Google Scholar]
  11. Chernomordik L. V., Frolov V. A., Leikina E., Bronk P., Zimmerberg J. The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. J Cell Biol. 1998 Mar 23;140(6):1369–1382. doi: 10.1083/jcb.140.6.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chernomordik L. V., Leikina E., Frolov V., Bronk P., Zimmerberg J. An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J Cell Biol. 1997 Jan 13;136(1):81–93. doi: 10.1083/jcb.136.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Danieli T., Pelletier S. L., Henis Y. I., White J. M. Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J Cell Biol. 1996 May;133(3):559–569. doi: 10.1083/jcb.133.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Düzgüneş N., Pedroso de Lima M. C., Stamatatos L., Flasher D., Alford D., Friend D. S., Nir S. Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells. J Gen Virol. 1992 Jan;73(Pt 1):27–37. doi: 10.1099/0022-1317-73-1-27. [DOI] [PubMed] [Google Scholar]
  16. Ellens H., Bentz J., Mason D., Zhang F., White J. M. Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density. Biochemistry. 1990 Oct 16;29(41):9697–9707. doi: 10.1021/bi00493a027. [DOI] [PubMed] [Google Scholar]
  17. Gutman O., Danieli T., White J. M., Henis Y. I. Effects of exposure to low pH on the lateral mobility of influenza hemagglutinin expressed at the cell surface: correlation between mobility inhibition and inactivation. Biochemistry. 1993 Jan 12;32(1):101–106. doi: 10.1021/bi00052a014. [DOI] [PubMed] [Google Scholar]
  18. Günther-Ausborn S., Schoen P., Bartoldus I., Wilschut J., Stegmann T. Role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity. J Virol. 2000 Mar;74(6):2714–2720. doi: 10.1128/jvi.74.6.2714-2720.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Han X., Bushweller J. H., Cafiso D. S., Tamm L. K. Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin. Nat Struct Biol. 2001 Aug;8(8):715–720. doi: 10.1038/90434. [DOI] [PubMed] [Google Scholar]
  20. Korte T., Epand R. F., Epand R. M., Blumenthal R. Role of the Glu residues of the influenza hemagglutinin fusion peptide in the pH dependence of fusion activity. Virology. 2001 Oct 25;289(2):353–361. doi: 10.1006/viro.2001.1108. [DOI] [PubMed] [Google Scholar]
  21. Korte T., Ludwig K., Booy F. P., Blumenthal R., Herrmann A. Conformational intermediates and fusion activity of influenza virus hemagglutinin. J Virol. 1999 Jun;73(6):4567–4574. doi: 10.1128/jvi.73.6.4567-4574.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Korte T., Ludwig K., Krumbiegel M., Zirwer D., Damaschun G., Herrmann A. Transient changes of the conformation of hemagglutinin of influenza virus at low pH detected by time-resolved circular dichroism spectroscopy. J Biol Chem. 1997 Apr 11;272(15):9764–9770. doi: 10.1074/jbc.272.15.9764. [DOI] [PubMed] [Google Scholar]
  23. Leikina E., LeDuc D. L., Macosko J. C., Epand R., Epand R., Shin Y. K., Chernomordik L. V. The 1-127 HA2 construct of influenza virus hemagglutinin induces cell-cell hemifusion. Biochemistry. 2001 Jul 27;40(28):8378–8386. doi: 10.1021/bi010466+. [DOI] [PubMed] [Google Scholar]
  24. Leikina E., Markovic I., Chernomordik L. V., Kozlov M. M. Delay of influenza hemagglutinin refolding into a fusion-competent conformation by receptor binding: a hypothesis. Biophys J. 2000 Sep;79(3):1415–1427. doi: 10.1016/S0006-3495(00)76393-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Markovic I., Leikina E., Zhukovsky M., Zimmerberg J., Chernomordik L. V. Synchronized activation and refolding of influenza hemagglutinin in multimeric fusion machines. J Cell Biol. 2001 Nov 26;155(5):833–844. doi: 10.1083/jcb.200103005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Melikyan G. B., Niles W. D., Cohen F. S. The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface. J Gen Physiol. 1995 Nov;106(5):783–802. doi: 10.1085/jgp.106.5.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mittal A., Bentz J. Comprehensive kinetic analysis of influenza hemagglutinin-mediated membrane fusion: role of sialate binding. Biophys J. 2001 Sep;81(3):1521–1535. doi: 10.1016/S0006-3495(01)75806-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mittal Aditya, Leikina Eugenia, Bentz Joe, Chernomordik Leonid V. Kinetics of influenza hemagglutinin-mediated membrane fusion as a function of technique. Anal Biochem. 2002 Apr 15;303(2):145–152. doi: 10.1006/abio.2002.5590. [DOI] [PubMed] [Google Scholar]
  29. Nir S., Düzgünes N., de Lima M. C., Hoekstra D. Fusion of enveloped viruses with cells and liposomes. Activity and inactivation. Cell Biophys. 1990 Oct;17(2):181–201. doi: 10.1007/BF02990496. [DOI] [PubMed] [Google Scholar]
  30. Puri A., Booy F. P., Doms R. W., White J. M., Blumenthal R. Conformational changes and fusion activity of influenza virus hemagglutinin of the H2 and H3 subtypes: effects of acid pretreatment. J Virol. 1990 Aug;64(8):3824–3832. doi: 10.1128/jvi.64.8.3824-3832.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Qiao H., Armstrong R. T., Melikyan G. B., Cohen F. S., White J. M. A specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype. Mol Biol Cell. 1999 Aug;10(8):2759–2769. doi: 10.1091/mbc.10.8.2759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ruigrok R. W., Andree P. J., Hooft van Huysduynen R. A., Mellema J. E. Characterization of three highly purified influenza virus strains by electron microscopy. J Gen Virol. 1984 Apr;65(Pt 4):799–802. doi: 10.1099/0022-1317-65-4-799. [DOI] [PubMed] [Google Scholar]
  33. Ruigrok R. W., Krijgsman P. C., de Ronde-Verloop F. M., de Jong J. C. Natural heterogeneity of shape, infectivity and protein composition in an influenza A (H3N2) virus preparation. Virus Res. 1985 Jul;3(1):69–76. doi: 10.1016/0168-1702(85)90042-5. [DOI] [PubMed] [Google Scholar]
  34. Shangguan T., Alford D., Bentz J. Influenza-virus-liposome lipid mixing is leaky and largely insensitive to the material properties of the target membrane. Biochemistry. 1996 Apr 16;35(15):4956–4965. doi: 10.1021/bi9526903. [DOI] [PubMed] [Google Scholar]
  35. Shangguan T., Siegel D. P., Lear J. D., Axelsen P. H., Alford D., Bentz J. Morphological changes and fusogenic activity of influenza virus hemagglutinin. Biophys J. 1998 Jan;74(1):54–62. doi: 10.1016/S0006-3495(98)77766-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Skehel J. J., Wiley D. C. Coiled coils in both intracellular vesicle and viral membrane fusion. Cell. 1998 Dec 23;95(7):871–874. doi: 10.1016/s0092-8674(00)81710-9. [DOI] [PubMed] [Google Scholar]
  37. Stegmann T., Morselt H. W., Booy F. P., van Breemen J. F., Scherphof G., Wilschut J. Functional reconstitution of influenza virus envelopes. EMBO J. 1987 Sep;6(9):2651–2659. doi: 10.1002/j.1460-2075.1987.tb02556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]

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