Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Dec;71(12):9427–9433. doi: 10.1128/jvi.71.12.9427-9433.1997

The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity.

E C Bos 1, W Luytjes 1, W J Spaan 1
PMCID: PMC230247  PMID: 9371603

Abstract

The spike protein (S) of the murine coronavirus mouse hepatitis virus strain A59 (MHV-A59) induces both virus-to-cell fusion during infection and syncytium formation. Thus far, only syncytium formation could be studied after transient expression of S. We have recently described a system in which viral infectivity is mimicked by using virus-like particles (VLPs) and reporter defective-interfering (DI) RNAs (E. C. W. Bos, W. Luytjes, H. Van der Meulen, H. K. Koerten, and W. J. M. Spaan, Virology 218:52-60, 1996). Production of VLPs of MHV-A59 was shown to be dependent on the expression of M and E. We now show in several ways that the infectivity of VLPs is dependent on S. Infectivity was lost when spikeless VLPs were produced. Infectivity was blocked upon treatment of the VLPs with MHV-A59-neutralizing anti-S monoclonal antibody (MAb) A2.3 but not with nonneutralizing anti-S MAb A1.4. When the target cells were incubated with antireceptor MAb CC1, which blocks MHV-A59 infection, VLPs did not infect the target cells. Thus, S-mediated VLP infectivity resembles MHV-A59 infectivity. The system can be used to identify domains in S that are essential for infectivity. As a first application, we investigated the requirements of cleavage of S for the infectivity of MHV-A59. We inserted three mutant S proteins that were previously shown to be uncleaved (E. C. W. Bos, L. Heijnen, W. Luytjes, and W. J. M. Spaan, Virology 214:453-463, 1995) into the VLPs. Here we show that cleavage of the spike protein of MHV-A59 is not required for infectivity.

Full Text

The Full Text of this article is available as a PDF (705.8 KB).

Selected References

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

  1. Blumberg B. M., Giorgi C., Rose K., Kolakofsky D. Sequence determination of the Sendai virus fusion protein gene. J Gen Virol. 1985 Feb;66(Pt 2):317–331. doi: 10.1099/0022-1317-66-2-317. [DOI] [PubMed] [Google Scholar]
  2. Bos E. C., Heijnen L., Luytjes W., Spaan W. J. Mutational analysis of the murine coronavirus spike protein: effect on cell-to-cell fusion. Virology. 1995 Dec 20;214(2):453–463. doi: 10.1006/viro.1995.0056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bos E. C., Luytjes W., van der Meulen H. V., Koerten H. K., Spaan W. J. The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. Virology. 1996 Apr 1;218(1):52–60. doi: 10.1006/viro.1996.0165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang R. Y., Hofmann M. A., Sethna P. B., Brian D. A. A cis-acting function for the coronavirus leader in defective interfering RNA replication. J Virol. 1994 Dec;68(12):8223–8231. doi: 10.1128/jvi.68.12.8223-8231.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Dveksler G. S., Pensiero M. N., Cardellichio C. B., Williams R. K., Jiang G. S., Holmes K. V., Dieffenbach C. W. Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV. J Virol. 1991 Dec;65(12):6881–6891. doi: 10.1128/jvi.65.12.6881-6891.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dveksler G. S., Pensiero M. N., Dieffenbach C. W., Cardellichio C. B., Basile A. A., Elia P. E., Holmes K. V. Mouse hepatitis virus strain A59 and blocking antireceptor monoclonal antibody bind to the N-terminal domain of cellular receptor. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1716–1720. doi: 10.1073/pnas.90.5.1716. [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. Freed E. O., Myers D. J., Risser R. Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4650–4654. doi: 10.1073/pnas.87.12.4650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gallagher T. M., Buchmeier M. J., Perlman S. Cell receptor-independent infection by a neurotropic murine coronavirus. Virology. 1992 Nov;191(1):517–522. doi: 10.1016/0042-6822(92)90223-C. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gallagher T. M., Escarmis C., Buchmeier M. J. Alteration of the pH dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein. J Virol. 1991 Apr;65(4):1916–1928. doi: 10.1128/jvi.65.4.1916-1928.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gallagher T. M. Murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein. J Virol. 1996 Jul;70(7):4683–4690. doi: 10.1128/jvi.70.7.4683-4690.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gallaher W. R. Detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus. Cell. 1987 Jul 31;50(3):327–328. doi: 10.1016/0092-8674(87)90485-5. [DOI] [PubMed] [Google Scholar]
  14. Garoff H., Frischauf A. M., Simons K., Lehrach H., Delius H. Nucleotide sequence of cdna coding for Semliki Forest virus membrane glycoproteins. Nature. 1980 Nov 20;288(5788):236–241. doi: 10.1038/288236a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Gilmore W., Fleming J. O., Stohlman S. A., Weiner L. P. Characterization of the structural proteins of the murine coronavirus strain A59 using monoclonal antibodies. Proc Soc Exp Biol Med. 1987 Jun;185(2):177–186. doi: 10.3181/00379727-185-42532. [DOI] [PubMed] [Google Scholar]
  17. Gombold J. L., Hingley S. T., Weiss S. R. Fusion-defective mutants of mouse hepatitis virus A59 contain a mutation in the spike protein cleavage signal. J Virol. 1993 Aug;67(8):4504–4512. doi: 10.1128/jvi.67.8.4504-4512.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grosse B., Siddell S. G. Single amino acid changes in the S2 subunit of the MHV surface glycoprotein confer resistance to neutralization by S1 subunit-specific monoclonal antibody. Virology. 1994 Aug 1;202(2):814–824. doi: 10.1006/viro.1994.1403. [DOI] [PubMed] [Google Scholar]
  19. Hernandez L. D., Hoffman L. R., Wolfsberg T. G., White J. M. Virus-cell and cell-cell fusion. Annu Rev Cell Dev Biol. 1996;12:627–661. doi: 10.1146/annurev.cellbio.12.1.627. [DOI] [PubMed] [Google Scholar]
  20. Hingley S. T., Gombold J. L., Lavi E., Weiss S. R. MHV-A59 fusion mutants are attenuated and display altered hepatotropism. Virology. 1994 Apr;200(1):1–10. doi: 10.1006/viro.1994.1156. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kim K. H., Narayanan K., Makino S. Assembled coronavirus from complementation of two defective interfering RNAs. J Virol. 1997 May;71(5):3922–3931. doi: 10.1128/jvi.71.5.3922-3931.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Luytjes W., Gerritsma H., Bos E., Spaan W. Characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain A59 with maturation defects in the spike protein. J Virol. 1997 Feb;71(2):949–955. doi: 10.1128/jvi.71.2.949-955.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Luytjes W., Sturman L. S., Bredenbeek P. J., Charite J., van der Zeijst B. A., Horzinek M. C., Spaan W. J. Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site. Virology. 1987 Dec;161(2):479–487. doi: 10.1016/0042-6822(87)90142-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Makino S., Lai M. M. High-frequency leader sequence switching during coronavirus defective interfering RNA replication. J Virol. 1989 Dec;63(12):5285–5292. doi: 10.1128/jvi.63.12.5285-5292.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Masters P. S., Koetzner C. A., Kerr C. A., Heo Y. Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus. J Virol. 1994 Jan;68(1):328–337. doi: 10.1128/jvi.68.1.328-337.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Meinkoth J., Wahl G. Hybridization of nucleic acids immobilized on solid supports. Anal Biochem. 1984 May 1;138(2):267–284. doi: 10.1016/0003-2697(84)90808-x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Opstelten D. J., de Groote P., Horzinek M. C., Vennema H., Rottier P. J. Disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins. J Virol. 1993 Dec;67(12):7394–7401. doi: 10.1128/jvi.67.12.7394-7401.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Peng D., Koetzner C. A., McMahon T., Zhu Y., Masters P. S. Construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins. J Virol. 1995 Sep;69(9):5475–5484. doi: 10.1128/jvi.69.9.5475-5484.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ricard C. S., Koetzner C. A., Sturman L. S., Masters P. S. A conditional-lethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions. Virus Res. 1995 Dec;39(2-3):261–276. doi: 10.1016/0168-1702(95)00100-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ricard C. S., Sturman L. S. Isolation of the subunits of the coronavirus envelope glycoprotein E2 by hydroxyapatite high-performance liquid chromatography. J Chromatogr. 1985 Jun 19;326:191–197. doi: 10.1016/S0021-9673(01)87445-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Schmidt M. F. Acylation of viral spike glycoproteins: a feature of enveloped RNA viruses. Virology. 1982 Jan 15;116(1):327–338. doi: 10.1016/0042-6822(82)90424-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Snijder E. J., Wassenaar A. L., Spaan W. J. Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. J Virol. 1994 Sep;68(9):5755–5764. doi: 10.1128/jvi.68.9.5755-5764.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Spaan W. J., Rottier P. J., Horzinek M. C., van der Zeijst B. A. Isolation and identification of virus-specific mRNAs in cells infected with mouse hepatitis virus (MHV-A59). Virology. 1981 Jan 30;108(2):424–434. doi: 10.1016/0042-6822(81)90449-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stauber R., Pfleiderera M., Siddell S. Proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for fusion activity. J Gen Virol. 1993 Feb;74(Pt 2):183–191. doi: 10.1099/0022-1317-74-2-183. [DOI] [PubMed] [Google Scholar]
  39. Sturman L. S., Ricard C. S., Holmes K. V. 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. J Virol. 1985 Dec;56(3):904–911. doi: 10.1128/jvi.56.3.904-911.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Taguchi F. Fusion formation by the uncleaved spike protein of murine coronavirus JHMV variant cl-2. J Virol. 1993 Mar;67(3):1195–1202. doi: 10.1128/jvi.67.3.1195-1202.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Taguchi F., Ikeda T., Shida H. Molecular cloning and expression of a spike protein of neurovirulent murine coronavirus JHMV variant cl-2. J Gen Virol. 1992 May;73(Pt 5):1065–1072. doi: 10.1099/0022-1317-73-5-1065. [DOI] [PubMed] [Google Scholar]
  42. Vennema H., Godeke G. J., Rossen J. W., Voorhout W. F., Horzinek M. C., Opstelten D. J., Rottier P. J. Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. EMBO J. 1996 Apr 15;15(8):2020–2028. doi: 10.1002/j.1460-2075.1996.tb00553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vennema H., Heijnen L., Zijderveld A., Horzinek M. C., Spaan W. J. Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly. J Virol. 1990 Jan;64(1):339–346. doi: 10.1128/jvi.64.1.339-346.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vennema H., Rijnbrand R., Heijnen L., Horzinek M. C., Spaan W. J. Enhancement of the vaccinia virus/phage T7 RNA polymerase expression system using encephalomyocarditis virus 5'-untranslated region sequences. Gene. 1991 Dec 15;108(2):201–209. doi: 10.1016/0378-1119(91)90435-E. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Williams R. K., Jiang G. S., Holmes K. V. Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5533–5536. doi: 10.1073/pnas.88.13.5533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Williams R. K., Jiang G. S., Snyder S. W., Frana M. F., Holmes K. V. Purification of the 110-kilodalton glycoprotein receptor for mouse hepatitis virus (MHV)-A59 from mouse liver and identification of a nonfunctional, homologous protein in MHV-resistant SJL/J mice. J Virol. 1990 Aug;64(8):3817–3823. doi: 10.1128/jvi.64.8.3817-3823.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yamada Y. K., Takimoto K., Yabe M., Taguchi F. Acquired fusion activity of a murine coronavirus MHV-2 variant with mutations in the proteolytic cleavage site and the signal sequence of the S protein. Virology. 1997 Jan 6;227(1):215–219. doi: 10.1006/viro.1996.8313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang L., Homberger F., Spaan W., Luytjes W. Recombinant genomic RNA of coronavirus MHV-A59 after coreplication with a DI RNA containing the MHV-RI spike gene. Virology. 1997 Mar 31;230(1):93–102. doi: 10.1006/viro.1997.8460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. van Berlo M. F., van den Brink W. J., Horzinek M. C., van der Zeijst B. A. Fatty acid acylation of viral proteins in murine hepatitis virus-infected cells. Brief report. Arch Virol. 1987;95(1-2):123–128. doi: 10.1007/BF01311339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. van der Most R. G., Bredenbeek P. J., Spaan W. J. A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs. J Virol. 1991 Jun;65(6):3219–3226. doi: 10.1128/jvi.65.6.3219-3226.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. van der Most R. G., Heijnen L., Spaan W. J., de Groot R. J. Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of coronavirus MHV-A59 via synthetic co-replicating RNAs. Nucleic Acids Res. 1992 Jul 11;20(13):3375–3381. doi: 10.1093/nar/20.13.3375. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES