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. 1994 Dec;67(6):2355–2360. doi: 10.1016/S0006-3495(94)80721-0

Kinetics of the low pH-induced conformational changes and fusogenic activity of influenza hemagglutinin.

M Krumbiegel 1, A Herrmann 1, R Blumenthal 1
PMCID: PMC1225619  PMID: 7696474

Abstract

The decrease of the intrinsic tryptophan fluorescence intensity of purified influenza (X31 strain) hemagglutinin (HA) was used to monitor the low pH-induced conformational change of this protein. The kinetics of the fluorescence decrease depended strongly on the pH. At pH optimal for fusion, the change in tryptophan fluorescence was fast and could be fitted to a monoexponential function. We measured a rate constant of 5.78 s-1 (t1/2 = 120 ms) at pH 4.9 using rapid stopped-flow mixing. Under suboptimal conditions (higher pH), the rate constant was decreased by an order of magnitude. In addition, a slow component appeared and the fluorescence decrease followed a sum of two exponentials. The kinetics of conformational changes were compared with those of the fusion of influenza virus with red blood cell membranes as assessed by the R18-dequenching assay. At optimal pH the HA conformational change was not rate-limiting for the fusion process. However, at sub-optimal pH, the slow transition to the fusogenic conformational of HA resulted in slower kinetics and decreased extent of fusion.

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Selected References

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

  1. Blumenthal R., Bali-Puri A., Walter A., Covell D., Eidelman O. pH-dependent fusion of vesicular stomatitis virus with Vero cells. Measurement by dequenching of octadecyl rhodamine fluorescence. J Biol Chem. 1987 Oct 5;262(28):13614–13619. [PubMed] [Google Scholar]
  2. Blumenthal R., Schoch C., Puri A., Clague M. J. A dissection of steps leading to viral envelope protein-mediated membrane fusion. Ann N Y Acad Sci. 1991;635:285–296. doi: 10.1111/j.1749-6632.1991.tb36499.x. [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. 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]
  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. Hoekstra D., de Boer T., Klappe K., Wilschut J. Fluorescence method for measuring the kinetics of fusion between biological membranes. Biochemistry. 1984 Nov 20;23(24):5675–5681. doi: 10.1021/bi00319a002. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Ruigrok R. W., Martin S. R., Wharton S. A., Skehel J. J., Bayley P. M., Wiley D. C. Conformational changes in the hemagglutinin of influenza virus which accompany heat-induced fusion of virus with liposomes. Virology. 1986 Dec;155(2):484–497. doi: 10.1016/0042-6822(86)90210-2. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Wharton S. A., Ruigrok R. W., Martin S. R., Skehel J. J., Bayley P. M., Weis W., Wiley D. C. Conformational aspects of the acid-induced fusion mechanism of influenza virus hemagglutinin. Circular dichroism and fluorescence studies. J Biol Chem. 1988 Mar 25;263(9):4474–4480. [PubMed] [Google Scholar]
  15. 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]
  16. 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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