Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Oct 1;123(1):57–65. doi: 10.1083/jcb.123.1.57

Cholesterol is required in the exit pathway of Semliki Forest virus

PMCID: PMC2119816  PMID: 8408205

Abstract

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

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

  1. Bron R., Wahlberg J. M., Garoff H., Wilschut J. Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein. EMBO J. 1993 Feb;12(2):693–701. doi: 10.1002/j.1460-2075.1993.tb05703.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. CLAYTON R. B. THE UTILIZATION OF STEROLS BY INSECTS. J Lipid Res. 1964 Jan;5:3–19. [PubMed] [Google Scholar]
  3. Dawidowicz E. A. Dynamics of membrane lipid metabolism and turnover. Annu Rev Biochem. 1987;56:43–61. doi: 10.1146/annurev.bi.56.070187.000355. [DOI] [PubMed] [Google Scholar]
  4. Eidelman O., Schlegel R., Tralka T. S., Blumenthal R. pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles. J Biol Chem. 1984 Apr 10;259(7):4622–4628. [PubMed] [Google Scholar]
  5. Froshauer S., Kartenbeck J., Helenius A. Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J Cell Biol. 1988 Dec;107(6 Pt 1):2075–2086. doi: 10.1083/jcb.107.6.2075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gaedigk-Nitschko K., Ding M. X., Levy M. A., Schlesinger M. J. Site-directed mutations in the Sindbis virus 6K protein reveal sites for fatty acylation and the underacylated protein affects virus release and virion structure. Virology. 1990 Mar;175(1):282–291. doi: 10.1016/0042-6822(90)90210-i. [DOI] [PubMed] [Google Scholar]
  7. Gaedigk-Nitschko K., Schlesinger M. J. The Sindbis virus 6K protein can be detected in virions and is acylated with fatty acids. Virology. 1990 Mar;175(1):274–281. doi: 10.1016/0042-6822(90)90209-a. [DOI] [PubMed] [Google Scholar]
  8. Gillies S., Stollar V. The production of high yields of infectious vesicular stomatitis virus in A. albopictus cells and comparisons with replication in BHK-21 cells. Virology. 1980 Dec;107(2):509–513. doi: 10.1016/0042-6822(80)90317-7. [DOI] [PubMed] [Google Scholar]
  9. Goldstein J. L., Brown M. S. Regulation of the mevalonate pathway. Nature. 1990 Feb 1;343(6257):425–430. doi: 10.1038/343425a0. [DOI] [PubMed] [Google Scholar]
  10. Hahn Y. S., Strauss E. G., Strauss J. H. Mapping of RNA- temperature-sensitive mutants of Sindbis virus: assignment of complementation groups A, B, and G to nonstructural proteins. J Virol. 1989 Jul;63(7):3142–3150. doi: 10.1128/jvi.63.7.3142-3150.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Igarashi A. Isolation of a Singh's Aedes albopictus cell clone sensitive to Dengue and Chikungunya viruses. J Gen Virol. 1978 Sep;40(3):531–544. doi: 10.1099/0022-1317-40-3-531. [DOI] [PubMed] [Google Scholar]
  12. Kail M., Hollinshead M., Ansorge W., Pepperkok R., Frank R., Griffiths G., Vaux D. The cytoplasmic domain of alphavirus E2 glycoprotein contains a short linear recognition signal required for viral budding. EMBO J. 1991 Sep;10(9):2343–2351. doi: 10.1002/j.1460-2075.1991.tb07773.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kielian M. C., Helenius A. Role of cholesterol in fusion of Semliki Forest virus with membranes. J Virol. 1984 Oct;52(1):281–283. doi: 10.1128/jvi.52.1.281-283.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kielian M. C., Keränen S., Käriäinen L., Helenius A. Membrane fusion mutants of Semliki Forest virus. J Cell Biol. 1984 Jan;98(1):139–145. doi: 10.1083/jcb.98.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kielian M., Helenius A. pH-induced alterations in the fusogenic spike protein of Semliki Forest virus. J Cell Biol. 1985 Dec;101(6):2284–2291. doi: 10.1083/jcb.101.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kielian M., Jungerwirth S., Sayad K. U., DeCandido S. Biosynthesis, maturation, and acid activation of the Semliki Forest virus fusion protein. J Virol. 1990 Oct;64(10):4614–4624. doi: 10.1128/jvi.64.10.4614-4624.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liljeström P., Garoff H. Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor. J Virol. 1991 Jan;65(1):147–154. doi: 10.1128/jvi.65.1.147-154.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liljeström P., Lusa S., Huylebroeck D., Garoff H. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol. 1991 Aug;65(8):4107–4113. doi: 10.1128/jvi.65.8.4107-4113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lobigs M., Wahlberg J. M., Garoff H. Spike protein oligomerization control of Semliki Forest virus fusion. J Virol. 1990 Oct;64(10):5214–5218. doi: 10.1128/jvi.64.10.5214-5218.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lusa S., Garoff H., Liljeström P. Fate of the 6K membrane protein of Semliki Forest virus during virus assembly. Virology. 1991 Dec;185(2):843–846. doi: 10.1016/0042-6822(91)90556-q. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Matlin K. S., Reggio H., Helenius A., Simons K. Pathway of vesicular stomatitis virus entry leading to infection. J Mol Biol. 1982 Apr 15;156(3):609–631. doi: 10.1016/0022-2836(82)90269-8. [DOI] [PubMed] [Google Scholar]
  23. Miller M. L., Brown D. T. Morphogenesis of Sindbis virus in three subclones of Aedes albopictus (mosquito) cells. J Virol. 1992 Jul;66(7):4180–4190. doi: 10.1128/jvi.66.7.4180-4190.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Naim H. Y., Koblet H. The cleavage of p62, the precursor of E2 and E3, is an early and continuous event in Semliki Forest virus-infected Aedes albopictus cells. Arch Virol. 1990;110(3-4):221–237. doi: 10.1007/BF01311290. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Ou J. H., Strauss E. G., Strauss J. H. Comparative studies of the 3'-terminal sequences of several alpha virus RNAs. Virology. 1981 Mar;109(2):281–289. doi: 10.1016/0042-6822(81)90499-2. [DOI] [PubMed] [Google Scholar]
  27. Pal R., Petri W. A., Jr, Wagner R. R. Alteration of the membrane lipid composition and infectivity of vesicular stomatitis virus by growth in a Chinese hamster ovary cell sterol mutant and in lipid-supplemented baby hamster kidney clone 21 cells. J Biol Chem. 1980 Aug 25;255(16):7688–7693. [PubMed] [Google Scholar]
  28. Pavan A., Covelli E., Pascale M. C., Lucania G., Bonatti S., Pinto da Silva P., Torrisi M. R. Dynamics of transmembrane proteins during Sindbis virus budding. J Cell Sci. 1992 May;102(Pt 1):149–155. doi: 10.1242/jcs.102.1.149. [DOI] [PubMed] [Google Scholar]
  29. Perez L., Guinea R., Carrasco L. Synthesis of Semliki Forest virus RNA requires continuous lipid synthesis. Virology. 1991 Jul;183(1):74–82. doi: 10.1016/0042-6822(91)90119-v. [DOI] [PubMed] [Google Scholar]
  30. Phalen T., Kielian M. Cholesterol is required for infection by Semliki Forest virus. J Cell Biol. 1991 Feb;112(4):615–623. doi: 10.1083/jcb.112.4.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Rothberg K. G., Heuser J. E., Donzell W. C., Ying Y. S., Glenney J. R., Anderson R. G. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. doi: 10.1016/0092-8674(92)90143-z. [DOI] [PubMed] [Google Scholar]
  33. Rothberg K. G., Ying Y. S., Kamen B. A., Anderson R. G. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J Cell Biol. 1990 Dec;111(6 Pt 2):2931–2938. doi: 10.1083/jcb.111.6.2931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simons K., Warren G. Semliki Forest virus: a probe for membrane traffic in the animal cell. Adv Protein Chem. 1984;36:79–132. doi: 10.1016/S0065-3233(08)60296-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stegmann T., Doms R. W., Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989;18:187–211. doi: 10.1146/annurev.bb.18.060189.001155. [DOI] [PubMed] [Google Scholar]
  36. Suomalainen M., Liljeström P., Garoff H. Spike protein-nucleocapsid interactions drive the budding of alphaviruses. J Virol. 1992 Aug;66(8):4737–4747. doi: 10.1128/jvi.66.8.4737-4747.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vogel R. H., Provencher S. W., von Bonsdorff C. H., Adrian M., Dubochet J. Envelope structure of Semliki Forest virus reconstructed from cryo-electron micrographs. Nature. 1986 Apr 10;320(6062):533–535. doi: 10.1038/320533a0. [DOI] [PubMed] [Google Scholar]
  38. Wahlberg J. M., Boere W. A., Garoff H. The heterodimeric association between the membrane proteins of Semliki Forest virus changes its sensitivity to low pH during virus maturation. J Virol. 1989 Dec;63(12):4991–4997. doi: 10.1128/jvi.63.12.4991-4997.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Wahlberg J. M., Garoff H. Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells. J Cell Biol. 1992 Jan;116(2):339–348. doi: 10.1083/jcb.116.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. White J., Helenius A. pH-dependent fusion between the Semliki Forest virus membrane and liposomes. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3273–3277. doi: 10.1073/pnas.77.6.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yeagle P. L. Cholesterol and the cell membrane. Biochim Biophys Acta. 1985 Dec 9;822(3-4):267–287. doi: 10.1016/0304-4157(85)90011-5. [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]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES