Home LiteratureArticle Details
PMID: 11500371 Published · ppublish English Journal Article

Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics.

The EMBO journal ·Vol. 20 ·No. 16 ·2001-08-15 ·Pages 4432-42

Cross KJ, Wharton SA, Skehel JJ, Wiley DC, Steinhauer DA

Abstract

Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.

MeSH Terms
Acids Animals Cell Line Cell Line, Transformed Cell Membrane/metabolism Cricetinae Dogs Gene Expression HeLa Cells Hemagglutinin Glycoproteins, Influenza Virus/chemistry,genetics,metabolism Humans Influenza A virus/genetics,growth & development,metabolism,physiology Membrane Fusion/physiology Mutagenesis Peptides/chemistry,genetics,metabolism Protein Conformation Protein Precursors/chemistry,genetics,metabolism
Chemicals
Acids Hemagglutinin Glycoproteins, Influenza Virus Peptides Protein Precursors
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Cross K J
National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Wharton S A
Skehel J J
Wiley D C
Steinhauer D A
References (62)
62 references, click to expand
  1. Structural characterization of the human respiratory syncytial virus fusion protein core.
    Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14172-7 PMID: 11106388
  2. The role of the membrane-spanning domain sequence in glycoprotein-mediated membrane fusion.
    Mol Biol Cell. 1999 Sep;10(9):2803-15 PMID: 10473628
  3. Activation of influenza A viruses by trypsin treatment.
    Virology. 1975 Dec;68(2):426-39 PMID: 173078
  4. Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide.
    Virology. 1975 Dec;68(2):440-54 PMID: 128196
  5. Two disulfide-linked polypeptide chains constitute the active F protein of paramyxoviruses.
    Virology. 1977 Jul 1;80(1):54-66 PMID: 195398
  6. Purification of the fusion protein of Sendai virus: analysis of the NH2-terminal sequence generated during precursor activation.
    Proc Natl Acad Sci U S A. 1978 Jun;75(6):2737-40 PMID: 208074
  7. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution.
    Nature. 1981 Jan 29;289(5796):366-73 PMID: 7464906
  8. Proteolytic activation of the influenza virus hemagglutinin: The structure of the cleavage site and the enzymes involved in cleavage.
    Virology. 1981 Dec;115(2):361-74 PMID: 7032055
  9. 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):968-72 PMID: 6951181
  10. Analyses of the antigenicity of influenza haemagglutinin at the pH optimum for virus-mediated membrane fusion.
    J Gen Virol. 1983 Aug;64 (Pt 8):1657-62 PMID: 6192202
  11. Sequential mutations in hemagglutinins of influenza B virus isolates: definition of antigenic domains.
    Proc Natl Acad Sci U S A. 1983 Jul;80(14):4527-31 PMID: 6192436
  12. Fusion mutants of the influenza virus hemagglutinin glycoprotein.
    Cell. 1985 Feb;40(2):431-9 PMID: 3967299
  13. 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 PMID: 3753607
  14. Studies of influenza haemagglutinin-mediated membrane fusion.
    Virology. 1986 Feb;149(1):27-35 PMID: 3946080
  15. Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway.
    J Cell Biol. 1986 Apr;102(4):1271-83 PMID: 3007532
  16. Conformational changes in the hemagglutinin of influenza virus which accompany heat-induced fusion of virus with liposomes.
    Virology. 1986 Dec;155(2):484-97 PMID: 3788061
  17. Membrane binding and conformational properties of peptides representing the NH2 terminus of influenza HA-2.
    J Biol Chem. 1987 May 15;262(14):6500-5 PMID: 3571268
  18. Membrane fusion by peptide analogues of influenza virus haemagglutinin.
    J Gen Virol. 1988 Aug;69 ( Pt 8):1847-57 PMID: 3404116
  19. Studies on the structure of the influenza virus haemagglutinin at the pH of membrane fusion.
    J Gen Virol. 1988 Nov;69 ( Pt 11):2785-95 PMID: 3183628
  20. Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence.
    J Biol Chem. 1989 Mar 5;264(7):3972-8 PMID: 2917985
  21. Delivery of macromolecules into cells expressing a viral membrane fusion protein.
    Methods Cell Biol. 1989;31:155-78 PMID: 2779447
  22. The structure of a membrane fusion mutant of the influenza virus haemagglutinin.
    EMBO J. 1990 Jan;9(1):17-24 PMID: 2295311
  23. Viral and cellular membrane fusion proteins.
    Annu Rev Physiol. 1990;52:675-97 PMID: 2184772
  24. Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses.
    Virology. 1991 Jun;182(2):475-85 PMID: 2024485
  25. Deacylation of the hemagglutinin of influenza A/Aichi/2/68 has no effect on membrane fusion properties.
    Virology. 1991 Sep;184(1):445-8 PMID: 1871979
  26. 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):11525-9 PMID: 1763066
  27. Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity.
    Cell. 1992 Feb 21;68(4):635-45 PMID: 1739972
  28. A chimeric avian retrovirus containing the influenza virus hemagglutinin gene has an expanded host range.
    J Virol. 1992 Dec;66(12):7374-82 PMID: 1331528
  29. 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):311-4 PMID: 8429306
  30. Mutations in the membrane-spanning domain of the human immunodeficiency virus envelope glycoprotein that affect fusion activity.
    J Virol. 1994 Jan;68(1):570-4 PMID: 8254774
  31. Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion.
    Cell. 1994 Jan 28;76(2):383-91 PMID: 8293471
  32. Structure of influenza haemagglutinin at the pH of membrane fusion.
    Nature. 1994 Sep 1;371(6492):37-43 PMID: 8072525
  33. Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin.
    J Virol. 1994 Nov;68(11):7537-9 PMID: 7933138
  34. Structure of influenza virus haemagglutinin complexed with a neutralizing antibody.
    Nature. 1995 Jul 6;376(6535):92-4 PMID: 7596443
  35. Selection of recombinant vaccinia viruses on the basis of plaque formation.
    Gene. 1995 Jun 9;158(2):157-62 PMID: 7607536
  36. Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin.
    J Virol. 1995 Nov;69(11):6643-51 PMID: 7474073
  37. Retrovirus envelope domain at 1.7 angstrom resolution.
    Nat Struct Biol. 1996 May;3(5):465-9 PMID: 8612078
  38. Glycoprotein incorporation and HIV-1 infectivity despite exchange of the gp160 membrane-spanning domain.
    Virology. 1996 Apr 1;218(1):269-74 PMID: 8615034
  39. Membrane fusion: the influenza paradigm.
    Cold Spring Harb Symp Quant Biol. 1995;60:581-8 PMID: 8824431
  40. Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers.
    Biophys J. 1996 May;70(5):2275-86 PMID: 9172751
  41. Atomic structure of the ectodomain from HIV-1 gp41.
    Nature. 1997 May 22;387(6631):426-30 PMID: 9163431
  42. Adaptation of egg-grown and transfectant influenza viruses for growth in mammalian cells: selection of hemagglutinin mutants with elevated pH of membrane fusion.
    Virology. 1997 Jul 7;233(2):402-10 PMID: 9217063
  43. Influence of membrane anchoring and cytoplasmic domains on the fusogenic activity of vesicular stomatitis virus glycoprotein G.
    J Virol. 1997 Oct;71(10):7996-8000 PMID: 9311894
  44. The transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus G protein.
    Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3425-30 PMID: 9520382
  45. The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil.
    Proc Natl Acad Sci U S A. 1998 May 26;95(11):6032-6 PMID: 9600912
  46. Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41.
    EMBO J. 1998 Aug 17;17(16):4572-84 PMID: 9707417
  47. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution.
    Nature. 1998 Sep 24;395(6700):347-53 PMID: 9759724
  48. Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation.
    Cell. 1998 Oct 30;95(3):409-17 PMID: 9814710
  49. Coiled coils in both intracellular vesicle and viral membrane fusion.
    Cell. 1998 Dec 23;95(7):871-4 PMID: 9875840
  50. Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-A resolution.
    Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2662-7 PMID: 10077567
  51. Structural basis for paramyxovirus-mediated membrane fusion.
    Mol Cell. 1999 Mar;3(3):309-19 PMID: 10198633
  52. Amino acid sequence requirements of the transmembrane and cytoplasmic domains of influenza virus hemagglutinin for viable membrane fusion.
    Mol Biol Cell. 1999 Jun;10(6):1821-36 PMID: 10359599
  53. N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil.
    Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):8967-72 PMID: 10430879
  54. Generation of influenza A viruses entirely from cloned cDNAs.
    Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9345-50 PMID: 10430945
  55. The lipid-anchored ectodomain of influenza virus hemagglutinin (GPI-HA) is capable of inducing nonenlarging fusion pores.
    Mol Biol Cell. 2000 Apr;11(4):1143-52 PMID: 10749920
  56. Structure of an analog of fusion peptide from hemagglutinin.
    Protein Sci. 2000 Apr;9(4):786-98 PMID: 10794422
  57. Electron microscopy of the human respiratory syncytial virus fusion protein and complexes that it forms with monoclonal antibodies.
    Virology. 2000 May 25;271(1):122-31 PMID: 10814577
  58. Modification of the cytoplasmic domain of influenza virus hemagglutinin affects enlargement of the fusion pore.
    J Virol. 2000 Aug;74(16):7529-37 PMID: 10906206
  59. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin.
    Annu Rev Biochem. 2000;69:531-69 PMID: 10966468
  60. Helical membrane protein folding, stability, and evolution.
    Annu Rev Biochem. 2000;69:881-922 PMID: 10966478
  61. A specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype.
    Mol Biol Cell. 1999 Aug;10(8):2759-69 PMID: 10436026
  62. Studies on the primary structure of the influenza virus hemagglutinin.
    Proc Natl Acad Sci U S A. 1975 Jan;72(1):93-7 PMID: 1054518
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/20.16.4432
Published
2001-08-15
Pages
4432-42
Language
English
Region
England
NLM ID
8208664
PMCID
PMC125568
Subset
IM
External Links
PubMed source DOI: 10.1093/emboj/20.16.4432 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2