Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1996 Feb;70(2):952–959. doi: 10.1128/jvi.70.2.952-959.1996

Events in the endoplasmic reticulum abrogate the temperature sensitivity of Sindbis virus mutant ts23.

M Carleton 1, D T Brown 1
PMCID: PMC189899  PMID: 8551635

Abstract

Temperature-sensitive mutations in proteins produced at or heated to a nonpermissive temperature render the proteins defective in some aspect of their maturation into functional entities. The characterization of temperature-sensitive mutations in model proteins, such as virus membrane proteins, has allowed the elucidation of critical events in the maturation of virus membranes as well as in the intracellular folding, processing, and transport of membrane proteins in general. We have used a transport-defective, temperature-sensitive mutant of Sindbis virus, ts23, which has two amino acid changes in the envelope protein E1, to further examine requirements placed upon the glycoproteins for their export to the plasma membrane. Pulse-chase experiments in which we utilized the transport inhibitors monensin and brefeldin A allowed us to synthesize and assemble the glycoproteins of ts23 into export-competent heterodimers at the permissive temperature while concurrently blocking their export to the cell surface. After removal of the inhibitors and a shift to the nonpermissive temperature, we assayed for protein transport, cell-cell fusion, and infectious-particle production. Taken together, the data show that the irreversible loss of the temperature-sensitive phenotype of ts23 can be correlated with the folding of E1 and the formation of export-competent PE2-E1 heterodimers in the endoplasmic reticulum. Furthermore, we have found that E1 pairs with PE2 to form the heterodimer prior to the completion of E1 folding.

Full Text

The Full Text of this article is available as a PDF (386.7 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. 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]
  3. 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]
  4. Burge B. W., Pfefferkorn E. R. Complementation between temperature-sensitive mutants of Sindbis virus. Virology. 1966 Oct;30(2):214–223. doi: 10.1016/0042-6822(66)90097-3. [DOI] [PubMed] [Google Scholar]
  5. Chege N. W., Pfeffer S. R. Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network. J Cell Biol. 1990 Sep;111(3):893–899. doi: 10.1083/jcb.111.3.893. [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. Doms R. W., Russ G., Yewdell J. W. Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J Cell Biol. 1989 Jul;109(1):61–72. doi: 10.1083/jcb.109.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubuisson J., Rice C. M. Sindbis virus attachment: isolation and characterization of mutants with impaired binding to vertebrate cells. J Virol. 1993 Jun;67(6):3363–3374. doi: 10.1128/jvi.67.6.3363-3374.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Erwin C., Brown D. T. Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants. J Virol. 1980 Dec;36(3):775–786. doi: 10.1128/jvi.36.3.775-786.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Griffiths G., Quinn P., Warren G. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J Cell Biol. 1983 Mar;96(3):835–850. doi: 10.1083/jcb.96.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Johnson D. C., Schlesinger M. J. Vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores. Virology. 1980 Jun;103(2):407–424. doi: 10.1016/0042-6822(80)90200-7. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Käriäinen L., Hashimoto K., Saraste J., Virtanen I., Penttinen K. Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins. J Cell Biol. 1980 Dec;87(3 Pt 1):783–791. doi: 10.1083/jcb.87.3.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Levy-Mintz P., Kielian M. Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein. J Virol. 1991 Aug;65(8):4292–4300. doi: 10.1128/jvi.65.8.4292-4300.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  20. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Lobigs M., Garoff H. Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62. J Virol. 1990 Mar;64(3):1233–1240. doi: 10.1128/jvi.64.3.1233-1240.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lobigs M., Zhao H. X., Garoff H. Function of Semliki Forest virus E3 peptide in virus assembly: replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1. J Virol. 1990 Sep;64(9):4346–4355. doi: 10.1128/jvi.64.9.4346-4355.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lustig S., Jackson A. C., Hahn C. S., Griffin D. E., Strauss E. G., Strauss J. H. Molecular basis of Sindbis virus neurovirulence in mice. J Virol. 1988 Jul;62(7):2329–2336. doi: 10.1128/jvi.62.7.2329-2336.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mann E., Edwards J., Brown D. T. Polycaryocyte formation mediated by Sindbis virus glycoproteins. J Virol. 1983 Mar;45(3):1083–1089. doi: 10.1128/jvi.45.3.1083-1089.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Migliaccio G., Pascale M. C., Leone A., Bonatti S. Biosynthesis, membrane translocation, and surface expression of Sindbis virus E1 glycoprotein. Exp Cell Res. 1989 Nov;185(1):203–216. doi: 10.1016/0014-4827(89)90049-9. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Olmsted R. A., Meyer W. J., Johnston R. E. Characterization of Sindbis virus epitopes important for penetration in cell culture and pathogenesis in animals. Virology. 1986 Jan 30;148(2):245–254. doi: 10.1016/0042-6822(86)90322-3. [DOI] [PubMed] [Google Scholar]
  32. Omar A., Koblet H. Semliki Forest virus particles containing only the E1 envelope glycoprotein are infectious and can induce cell-cell fusion. Virology. 1988 Sep;166(1):17–23. doi: 10.1016/0042-6822(88)90141-9. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. Rice C. M., Strauss J. H. Association of sindbis virion glycoproteins and their precursors. J Mol Biol. 1982 Jan 15;154(2):325–348. doi: 10.1016/0022-2836(82)90067-5. [DOI] [PubMed] [Google Scholar]
  38. Scheefers H., Scheefers-Borchel U., Edwards J., Brown D. T. Distribution of virus structural proteins and protein-protein interactions in plasma membrane of baby hamster kidney cells infected with Sindbis or vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7277–7281. doi: 10.1073/pnas.77.12.7277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smith J. F., Brown D. T. Envelopments of Sindbis virus: synthesis and organization of proteins in cells infected with wild type and maturation-defective mutants. J Virol. 1977 Jun;22(3):662–678. doi: 10.1128/jvi.22.3.662-678.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wahlberg J. M., Bron R., Wilschut J., Garoff H. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein. J Virol. 1992 Dec;66(12):7309–7318. doi: 10.1128/jvi.66.12.7309-7318.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yin F. H., Lockart R. Z., Jr Maturation defects in temperature-sensitive mutants of Sindbis virus. J Virol. 1968 Jul;2(7):728–737. doi: 10.1128/jvi.2.7.728-737.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ziemiecki A., Garoff H., Simons K. Formation of the Semliki Forest virus membrane glycoprotein complexes in the infected cell. J Gen Virol. 1980 Sep;50(1):111–123. doi: 10.1099/0022-1317-50-1-111. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. de Silva A. M., Balch W. E., Helenius A. Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro. J Cell Biol. 1990 Sep;111(3):857–866. doi: 10.1083/jcb.111.3.857. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES