Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 May;71(5):4062–4070. doi: 10.1128/jvi.71.5.4062-4070.1997

Molecular mechanism underlying the action of a novel fusion inhibitor of influenza A virus.

G Luo 1, A Torri 1, W E Harte 1, S Danetz 1, C Cianci 1, L Tiley 1, S Day 1, D Mullaney 1, K L Yu 1, C Ouellet 1, P Dextraze 1, N Meanwell 1, R Colonno 1, M Krystal 1
PMCID: PMC191559  PMID: 9094684

Abstract

In the initial stages of influenza virus infection, the hemagglutinin (HA) protein of influenza virus mediates both adsorption and penetration of the virus into the host cell. Recently, we identified and characterized BMY-27709 as an inhibitor of the H1 and H2 subtypes of influenza A virus that specifically inhibits the HA function necessary for virus-cell membrane fusion (G.-X. Luo, R. Colonno, and M. Krystal, Virology 226:66-76, 1996). Studies presented herein show that the inhibition is mediated through specific interaction with the HA protein. This binding represses the low-pH-induced conformational change of the HA protein which is a prerequisite for membrane fusion. In an attempt to define the binding pocket within the HA molecule, a number of drug-resistant viruses have been isolated and characterized. Sequence analyses of the HA gene of these drug-resistant viruses mapped amino acid changes responsible for drug resistance to a region located near the amino terminus of HA2. In addition, we have identified inactive analogs of BMY-27709 which are able to compete out the inhibitory activity of BMY-27709. This finding suggests that inhibition of the HA-mediated membrane fusion by this class of compounds is not solely the result of binding within the HA molecule but requires specific interactions.

Full Text

The Full Text of this article is available as a PDF (713.1 KB).

Selected References

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

  1. 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]
  2. Bodian D. L., Yamasaki R. B., Buswell R. L., Stearns J. F., White J. M., Kuntz I. D. Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones. Biochemistry. 1993 Mar 30;32(12):2967–2978. doi: 10.1021/bi00063a007. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Carr C. M., Kim P. S. A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell. 1993 May 21;73(4):823–832. doi: 10.1016/0092-8674(93)90260-w. [DOI] [PubMed] [Google Scholar]
  5. Carr C. M., Kim P. S. Flu virus invasion: halfway there. Science. 1994 Oct 14;266(5183):234–236. doi: 10.1126/science.7939658. [DOI] [PubMed] [Google Scholar]
  6. Clague M. J., Schoch C., Blumenthal R. Delay time for influenza virus hemagglutinin-induced membrane fusion depends on hemagglutinin surface density. J Virol. 1991 May;65(5):2402–2407. doi: 10.1128/jvi.65.5.2402-2407.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Durrer P., Galli C., Hoenke S., Corti C., Glück R., Vorherr T., Brunner J. H+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil region. J Biol Chem. 1996 Jun 7;271(23):13417–13421. doi: 10.1074/jbc.271.23.13417. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Gaudin Y., Ruigrok R. W., Brunner J. Low-pH induced conformational changes in viral fusion proteins: implications for the fusion mechanism. J Gen Virol. 1995 Jul;76(Pt 7):1541–1556. doi: 10.1099/0022-1317-76-7-1541. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Helenius A. Unpacking the incoming influenza virus. Cell. 1992 May 15;69(4):577–578. doi: 10.1016/0092-8674(92)90219-3. [DOI] [PubMed] [Google Scholar]
  16. Hiti A. L., Davis A. R., Nayak D. P. Complete sequence analysis shows that the hemagglutinins of the H0 and H2 subtypes of human influenza virus are closely related. Virology. 1981 May;111(1):113–124. doi: 10.1016/0042-6822(81)90658-9. [DOI] [PubMed] [Google Scholar]
  17. Hughson F. M. Structural characterization of viral fusion proteins. Curr Biol. 1995 Mar 1;5(3):265–274. doi: 10.1016/s0960-9822(95)00057-1. [DOI] [PubMed] [Google Scholar]
  18. Johnston R. F., Pickett S. C., Barker D. L. Autoradiography using storage phosphor technology. Electrophoresis. 1990 May;11(5):355–360. doi: 10.1002/elps.1150110503. [DOI] [PubMed] [Google Scholar]
  19. Kemble G. W., Danieli T., White J. M. Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell. 1994 Jan 28;76(2):383–391. doi: 10.1016/0092-8674(94)90344-1. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Luo G., Bergmann M., Garcia-Sastre A., Palese P. Mechanism of attenuation of a chimeric influenza A/B transfectant virus. J Virol. 1992 Aug;66(8):4679–4685. doi: 10.1128/jvi.66.8.4679-4685.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luo G., Colonno R., Krystal M. Characterization of a hemagglutinin-specific inhibitor of influenza A virus. Virology. 1996 Dec 1;226(1):66–76. doi: 10.1006/viro.1996.0628. [DOI] [PubMed] [Google Scholar]
  23. Melikyan G. B., Niles W. D., Peeples M. E., Cohen F. S. Influenza hemagglutinin-mediated fusion pores connecting cells to planar membranes: flickering to final expansion. J Gen Physiol. 1993 Dec;102(6):1131–1149. doi: 10.1085/jgp.102.6.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Morris S. J., Sarkar D. P., White J. M., Blumenthal R. 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):3972–3978. [PubMed] [Google Scholar]
  25. Nobusawa E., Aoyama T., Kato H., Suzuki Y., Tateno Y., Nakajima K. Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. Virology. 1991 Jun;182(2):475–485. doi: 10.1016/0042-6822(91)90588-3. [DOI] [PubMed] [Google Scholar]
  26. Pak C. C., Krumbiegel M., Blumenthal R. Intermediates in influenza virus PR/8 haemagglutinin-induced membrane fusion. J Gen Virol. 1994 Feb;75(Pt 2):395–399. doi: 10.1099/0022-1317-75-2-395. [DOI] [PubMed] [Google Scholar]
  27. Rafalski M., Ortiz A., Rockwell A., van Ginkel L. C., Lear J. D., DeGrado W. F., Wilschut J. Membrane fusion activity of the influenza virus hemagglutinin: interaction of HA2 N-terminal peptides with phospholipid vesicles. Biochemistry. 1991 Oct 22;30(42):10211–10220. doi: 10.1021/bi00106a020. [DOI] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sarkar D. P., Morris S. J., Eidelman O., Zimmerberg J., Blumenthal R. Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing. J Cell Biol. 1989 Jul;109(1):113–122. doi: 10.1083/jcb.109.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schoch C., Blumenthal R. Role of the fusion peptide sequence in initial stages of influenza hemagglutinin-induced cell fusion. J Biol Chem. 1993 May 5;268(13):9267–9274. [PubMed] [Google Scholar]
  31. 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]
  32. Spruce A. E., Iwata A., White J. M., Almers W. Patch clamp studies of single cell-fusion events mediated by a viral fusion protein. Nature. 1989 Nov 30;342(6249):555–558. doi: 10.1038/342555a0. [DOI] [PubMed] [Google Scholar]
  33. Stegmann T., Delfino J. M., Richards F. M., Helenius A. The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion. J Biol Chem. 1991 Sep 25;266(27):18404–18410. [PubMed] [Google Scholar]
  34. 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]
  35. Steinhauer D. A., Wharton S. A., Skehel J. J., Wiley D. C. Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin. J Virol. 1995 Nov;69(11):6643–6651. doi: 10.1128/jvi.69.11.6643-6651.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tatulian S. A., Hinterdorfer P., Baber G., Tamm L. K. Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy. EMBO J. 1995 Nov 15;14(22):5514–5523. doi: 10.1002/j.1460-2075.1995.tb00238.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tse F. W., Iwata A., Almers W. Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion. J Cell Biol. 1993 May;121(3):543–552. doi: 10.1083/jcb.121.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. White J. M. Membrane fusion. Science. 1992 Nov 6;258(5084):917–924. doi: 10.1126/science.1439803. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. White J. M., Wilson I. A. Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin. J Cell Biol. 1987 Dec;105(6 Pt 2):2887–2896. doi: 10.1083/jcb.105.6.2887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. 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]
  45. 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]
  46. Yewdell J. W., Gerhard W., Bachi T. Monoclonal anti-hemagglutinin antibodies detect irreversible antigenic alterations that coincide with the acid activation of influenza virus A/PR/834-mediated hemolysis. J Virol. 1983 Oct;48(1):239–248. doi: 10.1128/jvi.48.1.239-248.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yewdell J. W., Yellen A., Bächi T. Monoclonal antibodies localize events in the folding, assembly, and intracellular transport of the influenza virus hemagglutinin glycoprotein. Cell. 1988 Mar 25;52(6):843–852. doi: 10.1016/0092-8674(88)90426-6. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Zimmerberg J., Blumenthal R., Sarkar D. P., Curran M., Morris S. J. Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion. J Cell Biol. 1994 Dec;127(6 Pt 2):1885–1894. doi: 10.1083/jcb.127.6.1885. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES