Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1996 Aug;70(8):5541–5547. doi: 10.1128/jvi.70.8.5541-5547.1996

Disulfide bridge-mediated folding of Sindbis virus glycoproteins.

M Carleton 1, D T Brown 1
PMCID: PMC190513  PMID: 8764067

Abstract

The Sindbis virus envelope is composed of 80 E1-E2 (envelope glycoprotein) heterotrimers organized into an icosahedral protein lattice with T=4 symmetry. The structural integrity of the envelope protein lattice is maintained by E1-E1 interactions which are stabilized by intramolecular disulfide bonds. Structural domains of the envelope proteins sustain the envelope's icosahedral lattice, while functional domains are responsible for virus attachment and membrane fusion. We have previously shown that within the mature Sindbis virus particle, the structural domains of the envelope proteins are significantly more resistant to the membrane-permeative, sulfhydryl-reducing agent dithiothreitol (DTT) than are the functional domains (R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have used DTT to probe the accessibility of intramolecular disulfides within PE2 (the precursor to E2) and E1, as these proteins fold and are assembled into the spike heterotrimer. We have determined through pulse-chase analysis that intramolecular disulfide bonds within PE2 are always sensitive to DTT when the glycoproteins are in the endoplasmic reticulum. The reduction of these disulfides results in the disruption of PE2-E1 associations. E1 acquires increased resistance to DTT as it folds through a series of disulfide intermediates (E1alpha, -beta, and -gamma) prior to assuming its native and most compact conformation (E1epsilon). The transition from a DTT-sensitive form into a form which exhibits increased resistance to DTT occurs after E1 has folded into its E1beta conformation and correlates temporally with the dissociation of BiP-E1 complexes and the formation of PE2-E1 heterotrimers. We propose that the disulfide bonds within E1 which stabilize the protein domains required for maintaining the structural integrity of the envelope protein lattice form early within the folding pathway of E1 and become inaccessible to DTT once the heterotrimer has formed.

Full Text

The Full Text of this article is available as a PDF (512.2 KB).

Selected References

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

  1. Anthony R. P., Brown D. T. Protein-protein interactions in an alphavirus membrane. J Virol. 1991 Mar;65(3):1187–1194. doi: 10.1128/jvi.65.3.1187-1194.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anthony R. P., Paredes A. M., Brown D. T. Disulfide bonds are essential for the stability of the Sindbis virus envelope. Virology. 1992 Sep;190(1):330–336. doi: 10.1016/0042-6822(92)91219-k. [DOI] [PubMed] [Google Scholar]
  3. Barr P. J. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell. 1991 Jul 12;66(1):1–3. doi: 10.1016/0092-8674(91)90129-m. [DOI] [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Carleton M., Brown D. T. Events in the endoplasmic reticulum abrogate the temperature sensitivity of Sindbis virus mutant ts23. J Virol. 1996 Feb;70(2):952–959. doi: 10.1128/jvi.70.2.952-959.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coombs K., Brown D. T. Organization of the Sindbis virus nucleocapsid as revealed by bifunctional cross-linking agents. J Mol Biol. 1987 May 20;195(2):359–371. doi: 10.1016/0022-2836(87)90657-7. [DOI] [PubMed] [Google Scholar]
  7. Doms R. W., Lamb R. A., Rose J. K., Helenius A. Folding and assembly of viral membrane proteins. Virology. 1993 Apr;193(2):545–562. doi: 10.1006/viro.1993.1164. [DOI] [PubMed] [Google Scholar]
  8. Frolov I., Schlesinger S. Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells. J Virol. 1994 Mar;68(3):1721–1727. doi: 10.1128/jvi.68.3.1721-1727.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  10. Hashimoto K., Erdei S., Keränen S., Saraste J., Käriäinen L. Evidence for a separate signal sequence for the carboxy-terminal envelope glycoprotein E1 of Semliki forest virus. J Virol. 1981 Apr;38(1):34–40. doi: 10.1128/jvi.38.1.34-40.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hurtley S. M., Helenius A. Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:277–307. doi: 10.1146/annurev.cb.05.110189.001425. [DOI] [PubMed] [Google Scholar]
  12. Knipfer M. E., Brown D. T. Intracellular transport and processing of Sindbis virus glycoproteins. Virology. 1989 May;170(1):117–122. doi: 10.1016/0042-6822(89)90358-9. [DOI] [PubMed] [Google Scholar]
  13. Liu N., Brown D. T. Phosphorylation and dephosphorylation events play critical roles in Sindbis virus maturation. Virology. 1993 Oct;196(2):703–711. doi: 10.1006/viro.1993.1527. [DOI] [PubMed] [Google Scholar]
  14. Liu N., Brown D. T. Transient translocation of the cytoplasmic (endo) domain of a type I membrane glycoprotein into cellular membranes. J Cell Biol. 1993 Feb;120(4):877–883. doi: 10.1083/jcb.120.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Melancon P., Garoff H. Reinitiation of translocation in the Semliki Forest virus structural polyprotein: identification of the signal for the E1 glycoprotein. EMBO J. 1986 Jul;5(7):1551–1560. doi: 10.1002/j.1460-2075.1986.tb04396.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mendoza Q. P., Stanley J., Griffin D. E. Monoclonal antibodies to the E1 and E2 glycoproteins of Sindbis virus: definition of epitopes and efficiency of protection from fatal encephalitis. J Gen Virol. 1988 Dec;69(Pt 12):3015–3022. doi: 10.1099/0022-1317-69-12-3015. [DOI] [PubMed] [Google Scholar]
  17. Mulvey M., Brown D. T. Assembly of the Sindbis virus spike protein complex. Virology. 1996 May 1;219(1):125–132. doi: 10.1006/viro.1996.0229. [DOI] [PubMed] [Google Scholar]
  18. Mulvey M., Brown D. T. Formation and rearrangement of disulfide bonds during maturation of the Sindbis virus E1 glycoprotein. J Virol. 1994 Feb;68(2):805–812. doi: 10.1128/jvi.68.2.805-812.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mulvey M., Brown D. T. Involvement of the molecular chaperone BiP in maturation of Sindbis virus envelope glycoproteins. J Virol. 1995 Mar;69(3):1621–1627. doi: 10.1128/jvi.69.3.1621-1627.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Paredes A. M., Brown D. T., Rothnagel R., Chiu W., Schoepp R. J., Johnston R. E., Prasad B. V. Three-dimensional structure of a membrane-containing virus. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9095–9099. doi: 10.1073/pnas.90.19.9095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Paredes A. M., Simon M. N., Brown D. T. The mass of the Sindbis virus nucleocapsid suggests it has T = 4 icosahedral symmetry. Virology. 1992 Mar;187(1):329–332. doi: 10.1016/0042-6822(92)90322-g. [DOI] [PubMed] [Google Scholar]
  22. Presely J. F., Brown D. T. The proteolytic cleavage of PE2 to envelope glycoprotein E2 is not strictly required for the maturation of Sindbis virus. J Virol. 1989 May;63(5):1975–1980. doi: 10.1128/jvi.63.5.1975-1980.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Renz D., Brown D. T. Characteristics of Sindbis virus temperature-sensitive mutants in cultured BHK-21 and Aedes albopictus (Mosquito) cells. J Virol. 1976 Sep;19(3):775–781. doi: 10.1128/jvi.19.3.775-781.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Smith T. J., Cheng R. H., Olson N. H., Peterson P., Chase E., Kuhn R. J., Baker T. S. Putative receptor binding sites on alphaviruses as visualized by cryoelectron microscopy. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10648–10652. doi: 10.1073/pnas.92.23.10648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Strauss E. G., Stec D. S., Schmaljohn A. L., Strauss J. H. Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants. J Virol. 1991 Sep;65(9):4654–4664. doi: 10.1128/jvi.65.9.4654-4664.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tatu U., Braakman I., Helenius A. Membrane glycoprotein folding, oligomerization and intracellular transport: effects of dithiothreitol in living cells. EMBO J. 1993 May;12(5):2151–2157. doi: 10.1002/j.1460-2075.1993.tb05863.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tatu U., Hammond C., Helenius A. Folding and oligomerization of influenza hemagglutinin in the ER and the intermediate compartment. EMBO J. 1995 Apr 3;14(7):1340–1348. doi: 10.1002/j.1460-2075.1995.tb07120.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tucker P. C., Griffin D. E. Mechanism of altered Sindbis virus neurovirulence associated with a single-amino-acid change in the E2 Glycoprotein. J Virol. 1991 Mar;65(3):1551–1557. doi: 10.1128/jvi.65.3.1551-1557.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wang K. S., Schmaljohn A. L., Kuhn R. J., Strauss J. H. Antiidiotypic antibodies as probes for the Sindbis virus receptor. Virology. 1991 Apr;181(2):694–702. doi: 10.1016/0042-6822(91)90903-o. [DOI] [PubMed] [Google Scholar]
  31. Wang K. S., Strauss J. H. Use of a lambda gt11 expression library to localize a neutralizing antibody-binding site in glycoprotein E2 of Sindbis virus. J Virol. 1991 Dec;65(12):7037–7040. doi: 10.1128/jvi.65.12.7037-7040.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Watson D. G., Moehring J. M., Moehring T. J. A mutant CHO-K1 strain with resistance to Pseudomonas exotoxin A and alphaviruses fails to cleave Sindbis virus glycoprotein PE2. J Virol. 1991 May;65(5):2332–2339. doi: 10.1128/jvi.65.5.2332-2339.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. de Curtis I., Simons K. Dissection of Semliki Forest virus glycoprotein delivery from the trans-Golgi network to the cell surface in permeabilized BHK cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8052–8056. doi: 10.1073/pnas.85.21.8052. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES