Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1990 Oct;64(10):4884–4892. doi: 10.1128/jvi.64.10.4884-4892.1990

Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane.

R W Doms 1, R Blumenthal 1, B Moss 1
PMCID: PMC247978  PMID: 2398531

Abstract

The membrane fusion activities of the isolated single-envelope intracellular form of vaccinia virus (INV) and the double-envelope extracellular (EEV) form were studied by using a lipid-mixing assay based on the dilution of a fluorescent probe. Fluorescently labeled INV and EEV from both the IHD-J and WR strains of vaccinia virus fused with HeLa cells at neutral pH, suggesting that fusion occurs with the plasma membrane during virus entry. EEV fused more efficiently and with faster kinetics than INV: approximately 50% of bound EEV particles fused over the course of 1 h, compared with only 25% of the INV particles. Fusion of INV and EEV was strongly temperature dependent, being decreased by 50% at 34 degrees C and by 90% at 28 degrees C. A monoclonal antibody to a 14-kilodalton envelope protein of INV that has been implicated in the fusion reaction (J. F. Rodriguez, E. Paez, and M. Esteban, J. Virol. 61:395-404, 1987) completely suppressed the initial rate of fusion of INV but had no effect on the fusion activity of EEV, suggesting that vaccinia virus encodes two or more membrane fusion proteins. Finally, cells infected with the WR strain of vaccinia virus formed syncytia when briefly incubated at pH 6.4 or below, indicating that an acid-activated viral fusion protein is expressed on the cell surface. However, WR INV and EEV did not display increased fusion activity at acid pH, suggesting that the acid-dependent fusion factor is not incorporated into virions or that its activity there is masked.

Full text

PDF
4884

Images in this article

4889Image
on p.4889

Selected References

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

  1. Appleyard G., Hapel A. J., Boulter E. A. An antigenic difference between intracellular and extracellular rabbitpox virus. J Gen Virol. 1971 Oct;13(1):9–17. doi: 10.1099/0022-1317-13-1-9. [DOI] [PubMed] [Google Scholar]
  2. Armstrong J. A., Metz D. H., Young M. R. The mode of entry of vaccinia virus into L cells. J Gen Virol. 1973 Dec;21(3):533–537. doi: 10.1099/0022-1317-21-3-533. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Boulter E. A., Appleyard G. Differences between extracellular and intracellular forms of poxvirus and their implications. Prog Med Virol. 1973;16:86–108. [PubMed] [Google Scholar]
  5. Butcher M., Raviprakash K., Ghosh H. P. Acid pH-induced fusion of cells by herpes simplex virus glycoproteins gB an gD. J Biol Chem. 1990 Apr 5;265(10):5862–5868. [PubMed] [Google Scholar]
  6. Chang A., Metz D. H. Further investigations on the mode of entry of vaccinia virus into cells. J Gen Virol. 1976 Aug;32(2):275–282. doi: 10.1099/0022-1317-32-2-275. [DOI] [PubMed] [Google Scholar]
  7. Clague M. J., Schoch C., Zech L., Blumenthal R. Gating kinetics of pH-activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow measurements by dequenching of octadecylrhodamine fluorescence. Biochemistry. 1990 Feb 6;29(5):1303–1308. doi: 10.1021/bi00457a028. [DOI] [PubMed] [Google Scholar]
  8. DALES S., KAJIOKA R. THE CYCLE OF MULTIPLICATION OF VACCINIA VIRUS IN EARLE'S STRAIN L CELLS. I. UPTAKE AND PENETRATION. Virology. 1964 Nov;24:278–294. doi: 10.1016/0042-6822(64)90167-9. [DOI] [PubMed] [Google Scholar]
  9. Doms R. W., Gething M. J., Henneberry J., White J., Helenius A. Variant influenza virus hemagglutinin that induces fusion at elevated pH. J Virol. 1986 Feb;57(2):603–613. doi: 10.1128/jvi.57.2.603-613.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Florkiewicz R. Z., Rose J. K. A cell line expressing vesicular stomatitis virus glycoprotein fuses at low pH. Science. 1984 Aug 17;225(4663):721–723. doi: 10.1126/science.6087454. [DOI] [PubMed] [Google Scholar]
  11. Hiller G., Weber K. Golgi-derived membranes that contain an acylated viral polypeptide are used for vaccinia virus envelopment. J Virol. 1985 Sep;55(3):651–659. doi: 10.1128/jvi.55.3.651-659.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hoekstra D., Klappe K., de Boer T., Wilschut J. Characterization of the fusogenic properties of Sendai virus: kinetics of fusion with erythrocyte membranes. Biochemistry. 1985 Aug 27;24(18):4739–4745. doi: 10.1021/bi00339a005. [DOI] [PubMed] [Google Scholar]
  13. Hoekstra D., Kok J. W. Entry mechanisms of enveloped viruses. Implications for fusion of intracellular membranes. Biosci Rep. 1989 Jun;9(3):273–305. doi: 10.1007/BF01114682. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Ichihashi Y., Dales S. Biogenesis of poxviruses: interrelationship between hemagglutinin production and polykaryocytosis. Virology. 1971 Dec;46(3):533–543. doi: 10.1016/0042-6822(71)90057-2. [DOI] [PubMed] [Google Scholar]
  16. Ichihashi Y., Matsumoto S., Dales S. Biogenesis of poxviruses: role of A-type inclusions and host cell membranes in virus dissemination. Virology. 1971 Dec;46(3):507–532. doi: 10.1016/0042-6822(71)90056-0. [DOI] [PubMed] [Google Scholar]
  17. Ichihashi Y., Oie M. Adsorption and penetration of the trypsinized vaccinia virion. Virology. 1980 Feb;101(1):50–60. doi: 10.1016/0042-6822(80)90482-1. [DOI] [PubMed] [Google Scholar]
  18. Janeczko R. A., Rodriguez J. F., Esteban M. Studies on the mechanism of entry of vaccinia virus in animal cells. Arch Virol. 1987;92(1-2):135–150. doi: 10.1007/BF01310068. [DOI] [PubMed] [Google Scholar]
  19. Kondor-Koch C., Burke B., Garoff H. Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein. J Cell Biol. 1983 Sep;97(3):644–651. doi: 10.1083/jcb.97.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Maa J. S., Rodriguez J. F., Esteban M. Structural and functional characterization of a cell surface binding protein of vaccinia virus. J Biol Chem. 1990 Jan 25;265(3):1569–1577. [PubMed] [Google Scholar]
  22. Marsh M., Helenius A. Virus entry into animal cells. Adv Virus Res. 1989;36:107–151. doi: 10.1016/S0065-3527(08)60583-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Paterson R. G., Hiebert S. W., Lamb R. A. Expression at the cell surface of biologically active fusion and hemagglutinin/neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7520–7524. doi: 10.1073/pnas.82.22.7520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Payne L. G. Identification of the vaccinia hemagglutinin polypeptide from a cell system yielding large amounts of extracellular enveloped virus. J Virol. 1979 Jul;31(1):147–155. doi: 10.1128/jvi.31.1.147-155.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Payne L. G., Kristensson K. Extracellular release of enveloped vaccinia virus from mouse nasal epithelial cells in vivo. J Gen Virol. 1985 Mar;66(Pt 3):643–646. doi: 10.1099/0022-1317-66-3-643. [DOI] [PubMed] [Google Scholar]
  28. Payne L. G., Norrby E. Adsorption and penetration of enveloped and naked vaccinia virus particles. J Virol. 1978 Jul;27(1):19–27. doi: 10.1128/jvi.27.1.19-27.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Payne L. G., Norrby E. Presence of haemagglutinin in the envelope of extracellular vaccinia virus particles. J Gen Virol. 1976 Jul;32(1):63–72. doi: 10.1099/0022-1317-32-1-63. [DOI] [PubMed] [Google Scholar]
  30. Payne L. G. Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia. J Gen Virol. 1980 Sep;50(1):89–100. doi: 10.1099/0022-1317-50-1-89. [DOI] [PubMed] [Google Scholar]
  31. Payne L. Polypeptide composition of extracellular enveloped vaccinia virus. J Virol. 1978 Jul;27(1):28–37. doi: 10.1128/jvi.27.1.28-37.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Puri A., Winick J., Lowy R. J., Covell D., Eidelman O., Walter A., Blumenthal R. Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH. J Biol Chem. 1988 Apr 5;263(10):4749–4753. [PubMed] [Google Scholar]
  33. Riedel H., Kondor-Koch C., Garoff H. Cell surface expression of fusogenic vesicular stomatitis virus G protein from cloned cDNA. EMBO J. 1984 Jul;3(7):1477–1483. doi: 10.1002/j.1460-2075.1984.tb01999.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rodriguez J. F., Esteban M. Mapping and nucleotide sequence of the vaccinia virus gene that encodes a 14-kilodalton fusion protein. J Virol. 1987 Nov;61(11):3550–3554. doi: 10.1128/jvi.61.11.3550-3554.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rodriguez J. F., Janeczko R., Esteban M. Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus. J Virol. 1985 Nov;56(2):482–488. doi: 10.1128/jvi.56.2.482-488.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Seki M., Oie M., Ichihashi Y., Shida H. Hemadsorption and fusion inhibition activities of hemagglutinin analyzed by vaccinia virus mutants. Virology. 1990 Apr;175(2):372–384. doi: 10.1016/0042-6822(90)90422-n. [DOI] [PubMed] [Google Scholar]
  38. Shida H. Nucleotide sequence of the vaccinia virus hemagglutinin gene. Virology. 1986 Apr 30;150(2):451–462. doi: 10.1016/0042-6822(86)90309-0. [DOI] [PubMed] [Google Scholar]
  39. Stegmann T., Booy F. P., Wilschut J. Effects of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin. J Biol Chem. 1987 Dec 25;262(36):17744–17749. [PubMed] [Google Scholar]
  40. Stegmann T., Doms R. W., Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989;18:187–211. doi: 10.1146/annurev.bb.18.060189.001155. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Stern W., Dales S. Biogenesis of vaccinia: isolation and characterization of a surface component that elicits antibody suppressing infectivity and cell-cell fusion. Virology. 1976 Nov;75(1):232–241. doi: 10.1016/0042-6822(76)90022-2. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. 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]

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

RESOURCES