Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1985 Dec;56(3):904–911. doi: 10.1128/jvi.56.3.904-911.1985

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments.

L S Sturman, C S Ricard, K V Holmes
PMCID: PMC252663  PMID: 2999443

Abstract

In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell-fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities.

Full text

PDF
904

Images in this article

905Image
on p.905
906Image
on p.906

Selected References

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

  1. Bingham R. W., Madge M. H., Tyrrell D. A. Haemagglutination by avian infectious bronchitis virus-a coronavirus. J Gen Virol. 1975 Sep;28(3):381–390. doi: 10.1099/0022-1317-28-3-381. [DOI] [PubMed] [Google Scholar]
  2. Bosch F. X., Orlich M., Klenk H. D., Rott R. The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses. Virology. 1979 May;95(1):197–207. doi: 10.1016/0042-6822(79)90414-8. [DOI] [PubMed] [Google Scholar]
  3. CORBO L. J., CUNNINGHAM C. H. Hemagglutination by trypsin-modified infectious bronchitis virus. Am J Vet Res. 1959 Sep;20:876–883. [PubMed] [Google Scholar]
  4. Callebaut P. E., Pensaert M. B. Characterization and isolation of structural polypeptides in haemagglutinating encephalomyelitis virus. J Gen Virol. 1980 May;48(1):193–204. doi: 10.1099/0022-1317-48-1-193. [DOI] [PubMed] [Google Scholar]
  5. Cavanagh D. Coronavirus IBV: structural characterization of the spike protein. J Gen Virol. 1983 Dec;64(Pt 12):2577–2583. doi: 10.1099/0022-1317-64-12-2577. [DOI] [PubMed] [Google Scholar]
  6. Cavanagh D. Structural polypeptides of coronavirus IBV. J Gen Virol. 1981 Mar;53(Pt 1):93–103. doi: 10.1099/0022-1317-53-1-93. [DOI] [PubMed] [Google Scholar]
  7. Collins A. R., Knobler R. L., Powell H., Buchmeier M. J. Monoclonal antibodies to murine hepatitis virus-4 (strain JHM) define the viral glycoprotein responsible for attachment and cell--cell fusion. Virology. 1982 Jun;119(2):358–371. doi: 10.1016/0042-6822(82)90095-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frana M. F., Behnke J. N., Sturman L. S., Holmes K. V. Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion. J Virol. 1985 Dec;56(3):912–920. doi: 10.1128/jvi.56.3.912-920.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garten W., Berk W., Nagai Y., Rott R., Klenk H. D. Mutational changes of the protease susceptibility of glycoprotein F of Newcastle disease virus: effects on pathogenicity. J Gen Virol. 1980 Sep;50(1):135–147. doi: 10.1099/0022-1317-50-1-135. [DOI] [PubMed] [Google Scholar]
  10. Garwes D. J., Reynolds D. J. The polypeptide structure of canine coronavirus and its relationship to porcine transmissible gastroenteritis virus. J Gen Virol. 1981 Jan;52(Pt 1):153–157. doi: 10.1099/0022-1317-52-1-153. [DOI] [PubMed] [Google Scholar]
  11. Holmes K. V., Doller E. W., Behnke J. N. Analysis of the functions of coronavirus glycoproteins by differential inhibition of synthesis with tunicamycin. Adv Exp Med Biol. 1981;142:133–142. doi: 10.1007/978-1-4757-0456-3_11. [DOI] [PubMed] [Google Scholar]
  12. Holmes K. V., Doller E. W., Sturman L. S. Tunicamycin resistant glycosylation of coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein. Virology. 1981 Dec;115(2):334–344. doi: 10.1016/0042-6822(81)90115-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang R. T., Rott R., Klenk H. D. Influenza viruses cause hemolysis and fusion of cells. Virology. 1981 Apr 15;110(1):243–247. doi: 10.1016/0042-6822(81)90030-1. [DOI] [PubMed] [Google Scholar]
  14. Kaye H. S., Dowdle W. R. Some characteristics of hemagglutination of certain strains of "IBV-like" virus. J Infect Dis. 1969 Nov;120(5):576–581. doi: 10.1093/infdis/120.5.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. King B., Brian D. A. Bovine coronavirus structural proteins. J Virol. 1982 May;42(2):700–707. doi: 10.1128/jvi.42.2.700-707.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Klenk H. D., Rott R. Cotranslational and posttranslational processing of viral glycoproteins. Curr Top Microbiol Immunol. 1980;90:19–48. doi: 10.1007/978-3-642-67717-5_2. [DOI] [PubMed] [Google Scholar]
  17. Klenk H. D., Rott R., Orlich M., Blödorn J. Activation of influenza A viruses by trypsin treatment. Virology. 1975 Dec;68(2):426–439. doi: 10.1016/0042-6822(75)90284-6. [DOI] [PubMed] [Google Scholar]
  18. Lazarowitz S. G., Choppin P. W. Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology. 1975 Dec;68(2):440–454. doi: 10.1016/0042-6822(75)90285-8. [DOI] [PubMed] [Google Scholar]
  19. Lazarowitz S. G., Compans R. W., Choppin P. W. Proteolytic cleavage of the hemagglutinin polypeptide of influenza virus. Function of the uncleaved polypeptide HA. Virology. 1973 Mar;52(1):199–212. doi: 10.1016/0042-6822(73)90409-1. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Moss B., Rosenblum E. N. Hydroxylapatite chromatography of protein-sodium dodecyl sulfate complexes. A new method for the separation of polypeptide subunits. J Biol Chem. 1972 Aug 25;247(16):5194–5198. [PubMed] [Google Scholar]
  22. Nagai Y., Klenk H. D. Activation of precursors to both glycoporteins of Newcastle disease virus by proteolytic cleavage. Virology. 1977 Mar;77(1):125–134. doi: 10.1016/0042-6822(77)90412-3. [DOI] [PubMed] [Google Scholar]
  23. Nagai Y., Klenk H. D., Rott R. Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology. 1976 Jul 15;72(2):494–508. doi: 10.1016/0042-6822(76)90178-1. [DOI] [PubMed] [Google Scholar]
  24. Niemann H., Klenk H. D. Coronavirus glycoprotein E1, a new type of viral glycoprotein. J Mol Biol. 1981 Dec 25;153(4):993–1010. doi: 10.1016/0022-2836(81)90463-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Otsuki K., Tsubokura M. Plaque formation by avian infectious bronchitis virus in primary chick embryo fibroblast cells in the presence of trypsin. Arch Virol. 1981;70(4):315–320. doi: 10.1007/BF01320246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pocock D. H. Effect of sulphydryl reagents on the biological activities, polypeptide composition and morphology of haemagglutinating encephalomyelitis virus. J Gen Virol. 1978 Jul;40(1):93–101. doi: 10.1099/0022-1317-40-1-93. [DOI] [PubMed] [Google Scholar]
  27. Richardson C. D., Choppin P. W. Oligopeptides that specifically inhibit membrane fusion by paramyxoviruses: studies on the site of action. Virology. 1983 Dec;131(2):518–532. doi: 10.1016/0042-6822(83)90517-2. [DOI] [PubMed] [Google Scholar]
  28. Richardson C. D., Scheid A., Choppin P. W. Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides. Virology. 1980 Aug;105(1):205–222. doi: 10.1016/0042-6822(80)90168-3. [DOI] [PubMed] [Google Scholar]
  29. Rottier P. J., Horzinek M. C., van der Zeijst B. A. Viral protein synthesis in mouse hepatitis virus strain A59-infected cells: effect of tunicamycin. J Virol. 1981 Nov;40(2):350–357. doi: 10.1128/jvi.40.2.350-357.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rottier P. J., Spaan W. J., Horzinek M. C., van der Zeijst B. A. Translation of three mouse hepatitis virus strain A59 subgenomic RNAs in Xenopus laevis oocytes. J Virol. 1981 Apr;38(1):20–26. doi: 10.1128/jvi.38.1.20-26.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Scheid A., Choppin P. W. Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity of proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology. 1974 Feb;57(2):475–490. doi: 10.1016/0042-6822(74)90187-1. [DOI] [PubMed] [Google Scholar]
  32. Scheid A., Choppin P. W. Protease activation mutants of sendai virus. Activation of biological properties by specific proteases. Virology. 1976 Jan;69(1):265–277. doi: 10.1016/0042-6822(76)90213-0. [DOI] [PubMed] [Google Scholar]
  33. Siddell S., Wege H., Barthel A., ter Meulen V. Coronavirus JHM: intracellular protein synthesis. J Gen Virol. 1981 Mar;53(Pt 1):145–155. doi: 10.1099/0022-1317-53-1-145. [DOI] [PubMed] [Google Scholar]
  34. Siddell S., Wege H., Barthel A., ter Meulen V. Intracellular protein synthesis and the in vitro translation of coronavirus JHM mRNA. Adv Exp Med Biol. 1981;142:193–207. doi: 10.1007/978-1-4757-0456-3_17. [DOI] [PubMed] [Google Scholar]
  35. Stern D. F., Sefton B. M. Coronavirus proteins: biogenesis of avian infectious bronchitis virus virion proteins. J Virol. 1982 Dec;44(3):794–803. doi: 10.1128/jvi.44.3.794-803.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Storz J., Rott R., Kaluza G. Enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment. Infect Immun. 1981 Mar;31(3):1214–1222. doi: 10.1128/iai.31.3.1214-1222.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sturman L. S., Holmes K. V., Behnke J. Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid. J Virol. 1980 Jan;33(1):449–462. doi: 10.1128/jvi.33.1.449-462.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sturman L. S., Holmes K. V. Characterization of coronavirus II. Glycoproteins of the viral envelope: tryptic peptide analysis. Virology. 1977 Apr;77(2):650–660. doi: 10.1016/0042-6822(77)90489-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sturman L. S., Holmes K. V. Proteolytic cleavage of peplomeric glycoprotein E2 of MHV yields two 90K subunits and activates cell fusion. Adv Exp Med Biol. 1984;173:25–35. doi: 10.1007/978-1-4615-9373-7_3. [DOI] [PubMed] [Google Scholar]
  40. Sturman L. S., Holmes K. V. The molecular biology of coronaviruses. Adv Virus Res. 1983;28:35–112. doi: 10.1016/S0065-3527(08)60721-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sturman L. S. I. Structural proteins: effects of preparative conditions on the migration of protein in polyacrylamide gels. Virology. 1977 Apr;77(2):637–649. doi: 10.1016/0042-6822(77)90488-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sturman L. S., Takemoto K. K. Enhanced growth of a murine coronavirus in transformed mouse cells. Infect Immun. 1972 Oct;6(4):501–507. doi: 10.1128/iai.6.4.501-507.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sturman L. S. The structure and behavior of coronavirus A59 glycoproteins. Adv Exp Med Biol. 1981;142:1–17. doi: 10.1007/978-1-4757-0456-3_1. [DOI] [PubMed] [Google Scholar]
  44. Wege H., Dörries R., Wege H. Hybridoma antibodies to the murine coronavirus JHM: characterization of epitopes on the peplomer protein (E2). J Gen Virol. 1984 Nov;65(Pt 11):1931–1942. doi: 10.1099/0022-1317-65-11-1931. [DOI] [PubMed] [Google Scholar]
  45. Welch W. J., Sefton B. M. Two small virus-specific polypeptides are produced during infection with Sindbis virus. J Virol. 1979 Mar;29(3):1186–1195. doi: 10.1128/jvi.29.3.1186-1195.1979. [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]
  47. Yoshikura H., Tejima S. Role of protease in mouse hepatitis virus-induced cell fusion. Studies with a cold-sensitive mutant isolated from a persistent infection. Virology. 1981 Sep;113(2):503–511. doi: 10.1016/0042-6822(81)90178-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES