Folding of the mouse hepatitis virus spike protein and its association with the membrane protein

  • D.-J. E. Opstelten
  • P. de Groote
  • M. C. Horzinek
  • P. J. M. Rottier
Conference paper
Part of the Archives of Virology Supplementum book series (ARCHIVES SUPPL, volume 9)


Coronaviruses are assembled by budding into pre-Golgi membranes. Using different approaches we have demonstrated that the spike (S) protein and the membrane (M) protein of mouse hepatitis virus (MHV) associate to form large complexes. Newly synthesized M was found in these complexes almost immediately after its synthesis, whereas the S protein started to appear in heterocomplexes after 10–20 min. This is consistent with the slow rate of folding of S and with the observation that folding of S preceeds its association with M. While the folding of S involves the formation of multiple disulfide bonds, folding of M is disulfide-independent. This contrast was reflected by the differential sensitivity of the two proteins to reduction with dithiothreitol (DTT). Addition of DTT to the culture medium of MHV-infected cells drastically impaired the folding of S, but not of M. Consequently, the S protein was unable to interact with M. Under these conditions, S stayed in the ER while M was transported efficiently beyond the site of budding to the Golgi complex. We conclude that the association of S with M is an essential step in the formation of the viral envelope and in the accumulation of both proteins at the site of virus assembly.


Envelope Protein Golgi Complex Nucleocapsid Protein Disulfide Bond Formation Mouse Hepatitis Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Armstrong JH, Niemann H, Smeekens S, Rottier P, Warren G (1984) Sequence and topology of a model intracellular membrane protein, El glycoprotein, from a coronavirus. Nature 308: 751–752PubMedCrossRefGoogle Scholar
  2. 2.
    Braakman I, Hoover-Litty H, Wagner KR, Helenius A (1991) Folding of influenza hemagglutinin in the endoplasmic reticulum. J Cell Biol 114: 401–411PubMedCrossRefGoogle Scholar
  3. 3.
    Braakman I, Helenius J, Helenius A (1992) Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO J 11: 1717–1722Google Scholar
  4. 4.
    Griffiths G, Rottier P (1992) Cell biology of viruses that assemble along the biosynthetic pathway. Semin Cell Biol 3: 367–381PubMedCrossRefGoogle Scholar
  5. 5.
    Holmes KV, Doller EW, Sturman LS (1981) Tunicamycin resistant glycosylation of a coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein. Virology 115: 334–344PubMedCrossRefGoogle Scholar
  6. 6.
    Hurtley SM, Helenius A (1989) Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol 5: 277–307PubMedCrossRefGoogle Scholar
  7. 7.
    Krijnse Locker J, Griffiths G, Horzinek MC, Rottier PJM (1992) O-glycosylation of the Coronavirus M protein: differential localization of sialyltransferases in N- and O-linked glycosylation. J Biol Chem 267: 14094–14101PubMedGoogle Scholar
  8. 8.
    Pettersson R (1991) Protein localization and virus assembly at intracellular membranes. In: Compans RW (ed) Protein traffic in eukaryotic cells. Curr Top Microbiol Immunol 170: 67–106CrossRefGoogle Scholar
  9. 9.
    Rottier PJM, Horzinek MC, van der Zeijst BAM (1981) Viral protein synthesis in mouse hepatitis virus strain A59-infected cells: effects of tunicamycin. J Virol 40: 350–357PubMedGoogle Scholar
  10. 10.
    Rottier PJM, Welling GW, Welling-Wester S, Niesters HGM, Lenstra JA, van der Zeijst BAM (1986) Predicted membrane topology of the Coronavirus protein El. Biochemistry 25: 1335–1339PubMedCrossRefGoogle Scholar
  11. 11.
    Rottier PJM, Rose JK (1987) Coronavirus E1 glycoprotein expressed from cloned cDNA localizes in the Golgi region. J Virol 61: 2042–2045PubMedGoogle Scholar
  12. 12.
    Simons K, Garoff H (1980) The budding mechanism of enveloped animal viruses. J Gen Virol 50: 1–21PubMedCrossRefGoogle Scholar
  13. 13.
    Tooze J, Tooze S, Warren G (1984) Replication of Coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions. Eur J Cell Biol 33: 281–293PubMedGoogle Scholar
  14. 14.
    Tooze J, Tooze S, Warren G (1988) Site of addition of N-acetylgalactosamine to the E1 glycoprotein of mouse hepatitis virus-A59. J Cell Biol 106: 1475–1487PubMedCrossRefGoogle Scholar
  15. 15.
    Vennema H, Rottier PJM, Heijnen L, Godeke GJ, Horzinek MC, Spaan WJM (1990) Biosynthesis and function of the Coronavirus spike protein. In: Cavanagh D, Brown TDK (eds) Coronaviruses and their diseases. Plenum Press, New York, pp 9–19Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • D.-J. E. Opstelten
    • 1
  • P. de Groote
    • 1
  • M. C. Horzinek
    • 1
  • P. J. M. Rottier
    • 1
  1. 1.Institute of Virology, Department of Infectious Diseases and Immunology, Veterinary FacultyUtrecht UniversityThe Netherlands

Personalised recommendations