Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Nov;69(11):6643–6651. doi: 10.1128/jvi.69.11.6643-6651.1995

Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin.

D A Steinhauer 1, S A Wharton 1, J J Skehel 1, D C Wiley 1
PMCID: PMC189573  PMID: 7474073

Abstract

Influenza virus hemagglutinin (HA) fuses membranes at endosomal pH by a process which involves extrusion of the NH2-terminal region of HA2, the fusion peptide, from its buried location in the native trimer. We have examined the amino acid sequence requirements for a functional fusion peptide by determining the fusion capacities of site-specific mutant HAs expressed by using vaccinia virus recombinants and of synthetic peptide analogs of the mutant fusion peptides. The results indicate that for efficient fusion, alanine can to some extent substitute for the NH2-terminal glycine of the wild-type fusion peptide but that serine, histidine, leucine, isoleucine, or phenylalanine cannot. In addition, mutants containing shorter fusion peptides as a result of single amino acid deletions are inactive, as is a mutant containing an alanine instead of a glycine at HA2 residue 8. Substitution of the glycine at HA2 residue 4 with an alanine increases the pH of fusion, and valine-for-glutamate substitutions at HA2 residues 11 and 15 are without effect. We confirm previous reports on the need for specific HAo cleavage to generate functional HAs, and we show that both inappropriately cleaved HA and mutant HAs, irrespective of their fusion capacities, upon incubation at low pH undergo the structural transition required for fusion.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Bosch F. X., Garten W., Klenk H. D., Rott R. Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of Avian influenza viruses. Virology. 1981 Sep;113(2):725–735. doi: 10.1016/0042-6822(81)90201-4. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Daniels R. S., Douglas A. R., Skehel J. J., Wiley D. C. Analyses of the antigenicity of influenza haemagglutinin at the pH optimum for virus-mediated membrane fusion. J Gen Virol. 1983 Aug;64(Pt 8):1657–1662. doi: 10.1099/0022-1317-64-8-1657. [DOI] [PubMed] [Google Scholar]
  4. Daniels R. S., Downie J. C., Hay A. J., Knossow M., Skehel J. J., Wang M. L., Wiley D. C. Fusion mutants of the influenza virus hemagglutinin glycoprotein. Cell. 1985 Feb;40(2):431–439. doi: 10.1016/0092-8674(85)90157-6. [DOI] [PubMed] [Google Scholar]
  5. Garten W., Bosch F. X., Linder D., Rott R., Klenk H. D. Proteolytic activation of the influenza virus hemagglutinin: The structure of the cleavage site and the enzymes involved in cleavage. Virology. 1981 Dec;115(2):361–374. doi: 10.1016/0042-6822(81)90117-3. [DOI] [PubMed] [Google Scholar]
  6. Garten W., Klenk H. D. Characterization of the carboxypeptidase involved in the proteolytic cleavage of the influenza haemagglutinin. J Gen Virol. 1983 Oct;64(Pt 10):2127–2137. doi: 10.1099/0022-1317-64-10-2127. [DOI] [PubMed] [Google Scholar]
  7. Gething M. J., Doms R. W., York D., White J. Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus. J Cell Biol. 1986 Jan;102(1):11–23. doi: 10.1083/jcb.102.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Godley L., Pfeifer J., Steinhauer D., Ely B., Shaw G., Kaufmann R., Suchanek E., Pabo C., Skehel J. J., Wiley D. C. Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity. Cell. 1992 Feb 21;68(4):635–645. doi: 10.1016/0092-8674(92)90140-8. [DOI] [PubMed] [Google Scholar]
  9. Horimoto T., Nakayama K., Smeekens S. P., Kawaoka Y. Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses. J Virol. 1994 Sep;68(9):6074–6078. doi: 10.1128/jvi.68.9.6074-6078.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Huang R. T., Rott R., Klenk H. D. Influenza viruses cause hemolysis and fusion of cells. Virology. 1981 Apr 15;110(1):243–247. doi: 10.1016/0042-6822(81)90030-1. [DOI] [PubMed] [Google Scholar]
  11. Huang R. T., Wahn K., Klenk H. D., Rott R. Fusion between cell membrane and liposomes containing the glycoproteins of influenza virus. Virology. 1980 Jul 30;104(2):294–302. doi: 10.1016/0042-6822(80)90334-7. [DOI] [PubMed] [Google Scholar]
  12. Kido H., Yokogoshi Y., Sakai K., Tashiro M., Kishino Y., Fukutomi A., Katunuma N. Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein. J Biol Chem. 1992 Jul 5;267(19):13573–13579. [PubMed] [Google Scholar]
  13. Klenk H. D., Garten W. Host cell proteases controlling virus pathogenicity. Trends Microbiol. 1994 Feb;2(2):39–43. doi: 10.1016/0966-842x(94)90123-6. [DOI] [PubMed] [Google Scholar]
  14. Klenk H. D., Rott R., Orlich M., Blödorn J. Activation of influenza A viruses by trypsin treatment. Virology. 1975 Dec;68(2):426–439. doi: 10.1016/0042-6822(75)90284-6. [DOI] [PubMed] [Google Scholar]
  15. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  16. Lazarowitz S. G., Choppin P. W. Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology. 1975 Dec;68(2):440–454. doi: 10.1016/0042-6822(75)90285-8. [DOI] [PubMed] [Google Scholar]
  17. Lear J. D., DeGrado W. F. Membrane binding and conformational properties of peptides representing the NH2 terminus of influenza HA-2. J Biol Chem. 1987 May 15;262(14):6500–6505. [PubMed] [Google Scholar]
  18. Mackett M., Smith G. L., Moss B. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol. 1984 Mar;49(3):857–864. doi: 10.1128/jvi.49.3.857-864.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maeda T., Ohnishi S. Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes. FEBS Lett. 1980 Dec 29;122(2):283–287. doi: 10.1016/0014-5793(80)80457-1. [DOI] [PubMed] [Google Scholar]
  20. Murata M., Sugahara Y., Takahashi S., Ohnishi S. pH-dependent membrane fusion activity of a synthetic twenty amino acid peptide with the same sequence as that of the hydrophobic segment of influenza virus hemagglutinin. J Biochem. 1987 Oct;102(4):957–962. doi: 10.1093/oxfordjournals.jbchem.a122137. [DOI] [PubMed] [Google Scholar]
  21. Orlich M., Rott R. Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin. J Virol. 1994 Nov;68(11):7537–7539. doi: 10.1128/jvi.68.11.7537-7539.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Patel D. D., Ray C. A., Drucker R. P., Pickup D. J. A poxvirus-derived vector that directs high levels of expression of cloned genes in mammalian cells. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9431–9435. doi: 10.1073/pnas.85.24.9431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ruigrok R. W., Aitken A., Calder L. J., Martin S. R., Skehel J. J., Wharton S. A., Weis W., Wiley D. C. Studies on the structure of the influenza virus haemagglutinin at the pH of membrane fusion. J Gen Virol. 1988 Nov;69(Pt 11):2785–2795. doi: 10.1099/0022-1317-69-11-2785. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Scheiblauer H., Reinacher M., Tashiro M., Rott R. Interactions between bacteria and influenza A virus in the development of influenza pneumonia. J Infect Dis. 1992 Oct;166(4):783–791. doi: 10.1093/infdis/166.4.783. [DOI] [PubMed] [Google Scholar]
  26. Skehel J. J., Bayley P. M., Brown E. B., Martin S. R., Waterfield M. D., White J. M., Wilson I. A., Wiley D. C. Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc Natl Acad Sci U S A. 1982 Feb;79(4):968–972. doi: 10.1073/pnas.79.4.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Steinhauer D. A., Wharton S. A., Skehel J. J., Wiley D. C., Hay A. J. 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. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11525–11529. doi: 10.1073/pnas.88.24.11525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Steinhauer D. A., Wharton S. A., Wiley D. C., Skehel J. J. Deacylation of the hemagglutinin of influenza A/Aichi/2/68 has no effect on membrane fusion properties. Virology. 1991 Sep;184(1):445–448. doi: 10.1016/0042-6822(91)90867-b. [DOI] [PubMed] [Google Scholar]
  29. Stieneke-Gröber A., Vey M., Angliker H., Shaw E., Thomas G., Roberts C., Klenk H. D., Garten W. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J. 1992 Jul;11(7):2407–2414. doi: 10.1002/j.1460-2075.1992.tb05305.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tashiro M., Ciborowski P., Klenk H. D., Pulverer G., Rott R. Role of Staphylococcus protease in the development of influenza pneumonia. Nature. 1987 Feb 5;325(6104):536–537. doi: 10.1038/325536a0. [DOI] [PubMed] [Google Scholar]
  31. Venkatesan S., Baroudy B. M., Moss B. Distinctive nucleotide sequences adjacent to multiple initiation and termination sites of an early vaccinia virus gene. Cell. 1981 Sep;25(3):805–813. doi: 10.1016/0092-8674(81)90188-4. [DOI] [PubMed] [Google Scholar]
  32. Verhoeyen M., Fang R., Jou W. M., Devos R., Huylebroeck D., Saman E., Fiers W. Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/3/75. Nature. 1980 Aug 21;286(5775):771–776. doi: 10.1038/286771a0. [DOI] [PubMed] [Google Scholar]
  33. Walker J. A., Kawaoka Y. Importance of conserved amino acids at the cleavage site of the haemagglutinin of a virulent avian influenza A virus. J Gen Virol. 1993 Feb;74(Pt 2):311–314. doi: 10.1099/0022-1317-74-2-311. [DOI] [PubMed] [Google Scholar]
  34. Walker J. A., Molloy S. S., Thomas G., Sakaguchi T., Yoshida T., Chambers T. M., Kawaoka Y. Sequence specificity of furin, a proprotein-processing endoprotease, for the hemagglutinin of a virulent avian influenza virus. J Virol. 1994 Feb;68(2):1213–1218. doi: 10.1128/jvi.68.2.1213-1218.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wharton S. A., Martin S. R., Ruigrok R. W., Skehel J. J., Wiley D. C. Membrane fusion by peptide analogues of influenza virus haemagglutinin. J Gen Virol. 1988 Aug;69(Pt 8):1847–1857. doi: 10.1099/0022-1317-69-8-1847. [DOI] [PubMed] [Google Scholar]
  36. White J., Kielian M., Helenius A. Membrane fusion proteins of enveloped animal viruses. Q Rev Biophys. 1983 May;16(2):151–195. doi: 10.1017/s0033583500005072. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. 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]

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

RESOURCES