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. 1991 Aug;65(8):4063–4069. doi: 10.1128/jvi.65.8.4063-4069.1991

Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay.

N Srinivasakumar 1, P L Ogra 1, T D Flanagan 1
PMCID: PMC248838  PMID: 1906550

Abstract

The characteristics of fusion of respiratory syncytial virus (RSV) with HEp-2 cells were studied by the R18 fluorescence dequenching assay of membrane fusion. A gradual increase in fluorescence intensity indicative of virion-cell fusion was observed when R18-labeled RSV was incubated with HEp-2 cells. Approximately 35% dequenching of the probe fluorescence was observed in 1 h at 37 degrees C. Fusion showed a temperature dependence, with significant dequenching occurring above 18 degrees C. The dequenching was also dependent on the relative concentration of target membrane. Thus, increasing the concentration of target membrane resulted in increased levels of dequenching. In addition, viral glycoproteins were shown to be involved in this interaction, since dequenching was significantly reduced by pretreatment of labeled virus at 70 degrees C for 5 min or by trypsinization of R18-labeled virions prior to incubation with HEp-2 cells at 37 degrees C. The fusion of RSV with HEp-2 cells was unaffected over a pH range of 5.5 to 8.5, with some increase seen at lower pH values. Treatment of HEp-2 cells with ammonium chloride (20 and 10 mM), a lysosomotropic agent, during early stages of infection did not inhibit syncytium formation or progeny virion production by RSV. At the same concentrations of ammonium chloride, the production of vesicular stomatitis virus was reduced approximately 4 log10 units. These results suggest that fusion of the virus with the cell surface plasma membrane is the principal route of entry.

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Selected References

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  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. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Chejanovsky N., Henis Y. I., Loyter A. Fusion of fluorescently labeled Sendai virus envelopes with living cultured cells as monitored by fluorescence dequenching. Exp Cell Res. 1986 Jun;164(2):353–365. doi: 10.1016/0014-4827(86)90034-0. [DOI] [PubMed] [Google Scholar]
  4. Collins P. L., Huang Y. T., Wertz G. W. Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes. J Virol. 1984 Feb;49(2):572–578. doi: 10.1128/jvi.49.2.572-578.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collins P. L., Olmsted R. A., Johnson P. R. The small hydrophobic protein of human respiratory syncytial virus: comparison between antigenic subgroups A and B. J Gen Virol. 1990 Jul;71(Pt 7):1571–1576. doi: 10.1099/0022-1317-71-7-1571. [DOI] [PubMed] [Google Scholar]
  6. Doms R. W., Blumenthal R., Moss B. Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane. J Virol. 1990 Oct;64(10):4884–4892. doi: 10.1128/jvi.64.10.4884-4892.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elango N., Satake M., Coligan J. E., Norrby E., Camargo E., Venkatesan S. Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit. Nucleic Acids Res. 1985 Mar 11;13(5):1559–1574. doi: 10.1093/nar/13.5.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Gibson S., Bundo-Morita K., Portner A., Lenard J. Fusion of a Sendai mutant deficient in HN protein (ts271) with cardiolipin liposomes. Virology. 1988 Mar;163(1):226–229. doi: 10.1016/0042-6822(88)90254-1. [DOI] [PubMed] [Google Scholar]
  10. Gilbert J. M., Mason D., White J. M. Fusion of Rous sarcoma virus with host cells does not require exposure to low pH. J Virol. 1990 Oct;64(10):5106–5113. doi: 10.1128/jvi.64.10.5106-5113.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gimenez H. B., Keir H. M., Cash P. In vitro enhancement of respiratory syncytial virus infection of U937 cells by human sera. J Gen Virol. 1989 Jan;70(Pt 1):89–96. doi: 10.1099/0022-1317-70-1-89. [DOI] [PubMed] [Google Scholar]
  12. Haddad R. S., Hutt-Fletcher L. M. Depletion of glycoprotein gp85 from virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with virus receptor-bearing cells. J Virol. 1989 Dec;63(12):4998–5005. doi: 10.1128/jvi.63.12.4998-5005.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harmsen M. C., Wilschut J., Scherphof G., Hulstaert C., Hoekstra D. Reconstitution and fusogenic properties of Sendai virus envelopes. Eur J Biochem. 1985 Jun 18;149(3):591–599. doi: 10.1111/j.1432-1033.1985.tb08966.x. [DOI] [PubMed] [Google Scholar]
  14. Haywood A. M., Boyer B. P. Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration. Biochemistry. 1982 Nov 23;21(24):6041–6046. doi: 10.1021/bi00267a003. [DOI] [PubMed] [Google Scholar]
  15. Henis Y. I., Herman-Barhom Y., Aroeti B., Gutman O. Lateral mobility of both envelope proteins (F and HN) of Sendai virus in the cell membrane is essential for cell-cell fusion. J Biol Chem. 1989 Oct 15;264(29):17119–17125. [PubMed] [Google Scholar]
  16. Hoekstra D., Klappe K., Hoff H., Nir S. Mechanism of fusion of Sendai virus: role of hydrophobic interactions and mobility constraints of viral membrane proteins. Effects of polyethylene glycol. J Biol Chem. 1989 Apr 25;264(12):6786–6792. [PubMed] [Google Scholar]
  17. Hoekstra D., Klappe K. Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion. J Virol. 1986 Apr;58(1):87–95. doi: 10.1128/jvi.58.1.87-95.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Huang Y. T., Collins P. L., Wertz G. W. Characterization of the 10 proteins of human respiratory syncytial virus: identification of a fourth envelope-associated protein. Virus Res. 1985 Mar;2(2):157–173. doi: 10.1016/0168-1702(85)90246-1. [DOI] [PubMed] [Google Scholar]
  20. Kim H. W., Canchola J. G., Brandt C. D., Pyles G., Chanock R. M., Jensen K., Parrott R. H. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol. 1969 Apr;89(4):422–434. doi: 10.1093/oxfordjournals.aje.a120955. [DOI] [PubMed] [Google Scholar]
  21. Klenk H. D., Choppin P. W. Chemical composition of the parainfluenza virus SV5. Virology. 1969 Jan;37(1):155–157. doi: 10.1016/0042-6822(69)90321-3. [DOI] [PubMed] [Google Scholar]
  22. Krilov L. R., Anderson L. J., Marcoux L., Bonagura V. R., Wedgwood J. F. Antibody-mediated enhancement of respiratory syncytial virus infection in two monocyte/macrophage cell lines. J Infect Dis. 1989 Nov;160(5):777–782. doi: 10.1093/infdis/160.5.777. [DOI] [PubMed] [Google Scholar]
  23. Levine S., Klaiber-Franco R., Paradiso P. R. Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus. J Gen Virol. 1987 Sep;68(Pt 9):2521–2524. doi: 10.1099/0022-1317-68-9-2521. [DOI] [PubMed] [Google Scholar]
  24. Loyter A., Citovsky V., Blumenthal R. The use of fluorescence dequenching measurements to follow viral membrane fusion events. Methods Biochem Anal. 1988;33:129–164. doi: 10.1002/9780470110546.ch4. [DOI] [PubMed] [Google Scholar]
  25. Maeda T., Kawasaki K., Ohnishi S. Interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at pH 5.2. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4133–4137. doi: 10.1073/pnas.78.7.4133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Metsikkö K., van Meer G., Simons K. Reconstitution of the fusogenic activity of vesicular stomatitis virus. EMBO J. 1986 Dec 20;5(13):3429–3435. doi: 10.1002/j.1460-2075.1986.tb04665.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Miller N., Hutt-Fletcher L. M. A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus. J Virol. 1988 Jul;62(7):2366–2372. doi: 10.1128/jvi.62.7.2366-2372.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nussbaum O., Lapidot M., Loyter A. Reconstitution of functional influenza virus envelopes and fusion with membranes and liposomes lacking virus receptors. J Virol. 1987 Jul;61(7):2245–2252. doi: 10.1128/jvi.61.7.2245-2252.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nussbaum O., Loyter A. Quantitative determination of virus-membrane fusion events. Fusion of influenza virions with plasma membranes and membranes of endocytic vesicles in living cultured cells. FEBS Lett. 1987 Aug 31;221(1):61–67. doi: 10.1016/0014-5793(87)80352-6. [DOI] [PubMed] [Google Scholar]
  30. Routledge E. G., Willcocks M. M., Morgan L., Samson A. C., Scott R., Toms G. L. Expression of the respiratory syncytial virus 22K protein on the surface of infected HeLa cells. J Gen Virol. 1987 Apr;68(Pt 4):1217–1222. doi: 10.1099/0022-1317-68-4-1217. [DOI] [PubMed] [Google Scholar]
  31. Scheule R. K. Fusion of Sindbis virus with model membranes containing phosphatidylethanolamine: implications for protein-induced membrane fusion. Biochim Biophys Acta. 1987 May 29;899(2):185–195. doi: 10.1016/0005-2736(87)90399-3. [DOI] [PubMed] [Google Scholar]
  32. Stegmann T., Morselt H. W., Scholma J., Wilschut J. Fusion of influenza virus in an intracellular acidic compartment measured by fluorescence dequenching. Biochim Biophys Acta. 1987 Nov 2;904(1):165–170. doi: 10.1016/0005-2736(87)90100-3. [DOI] [PubMed] [Google Scholar]
  33. Struck D. K., Hoekstra D., Pagano R. E. Use of resonance energy transfer to monitor membrane fusion. Biochemistry. 1981 Jul 7;20(14):4093–4099. doi: 10.1021/bi00517a023. [DOI] [PubMed] [Google Scholar]
  34. Svensson U., Persson R. Entry of adenovirus 2 into HeLa cells. J Virol. 1984 Sep;51(3):687–694. doi: 10.1128/jvi.51.3.687-694.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tsutsumi H., Flanagan T. D., Ogra P. L. Monoclonal antibodies to the large glycoproteins of respiratory syncytial virus: possible evidence for several functional antigenic sites. J Gen Virol. 1987 Aug;68(Pt 8):2161–2167. doi: 10.1099/0022-1317-68-8-2161. [DOI] [PubMed] [Google Scholar]
  36. Ueba O. Respiratory syncytial virus. I. Concentration and purification of the infectious virus. Acta Med Okayama. 1978 Aug;32(4):265–272. [PubMed] [Google Scholar]

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