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. 1995 Jan;69(1):469–475. doi: 10.1128/jvi.69.1.469-475.1995

The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process.

A Loewy 1, J Smyth 1, C H von Bonsdorff 1, P Liljeström 1, M J Schlesinger 1
PMCID: PMC188595  PMID: 7983743

Abstract

Alphavirus genomes encode a small hydrophobic protein of 6 kDa (the 6K protein) that is expressed as part of a large polyprotein containing the sequences of the two virus transmembranal glycoproteins which form the spikes of the infectious particle. Although made in amounts equivalent to those of the glycoproteins, very little of the 6K protein is found in secreted infectious virions. The role of this protein in virus replication and structure has been studied by use of a variety of mutationally altered forms of 6K, which yield phenotypically distinct viruses. A complete deletion of the gene encoding the 6K protein (delta 6K) of Semliki Forest Virus (SFV) has been constructed from an SFV infectious cDNA and the transcribed RNA-produced progeny virus that closely resembled the normal virus (P. Liljeström, S. Lusa, D. Huylebroeck, and H. Garoff, J. Virol. 65:4107-4113, 1991). Further studies of this mutant have now been performed, and they show that growth of delta 6K has a strong dependency on its host cell, varying from 2 to 50% of the rate of formation of the wild-type SFV. Mammalian cells are much more defective than insect and avian cells in replication of the delta 6K mutant. This mutant is not defective in formation and transport of the glycoproteins or in production of nucleocapsids, which accumulate at the plasma cell membrane in infected BHK cells. The major defect, thus, is in the final assembly and budding of new virus. In BHK cells infected with the delta 6K strain, a relatively large fraction of the total infectious virus formed can be recovered by osmotic lysis of exhaustively washed cells. Infectious SFV totally lacking 6K is identical to wild-type SFV in the early stages of virus replication, i.e., binding and uptake. The particles themselves are more thermolabile than those of wild-type SFV, suggesting that the 6K protein may be a part of the structure of wild-type virus or that the slower budding leads to an altered configuration of the trimeric spikes. These data support other studies that implicate the 6K protein as an important but nonessential component in the assembly and budding of the alphavirus particle, perhaps by affecting the packing of the glycoproteins and their interactions with membrane lipid.

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Selected References

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  1. Aloia R. C., Tian H., Jensen F. C. Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5181–5185. doi: 10.1073/pnas.90.11.5181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Boere W. A., Harmsen T., Vinjé J., Benaissa-Trouw B. J., Kraaijeveld C. A., Snippe H. Identification of distinct antigenic determinants on Semliki Forest virus by using monoclonal antibodies with different antiviral activities. J Virol. 1984 Nov;52(2):575–582. doi: 10.1128/jvi.52.2.575-582.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown D. T., Smith J. F. Morphology of BHK-21 Cells Infected with Sindbis Virus Temperature-Sensitive Mutants in Complementation Groups D and E. J Virol. 1975 May;15(5):1262–1266. doi: 10.1128/jvi.15.5.1262-1266.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ekström M., Liljeström P., Garoff H. Membrane protein lateral interactions control Semliki Forest virus budding. EMBO J. 1994 Mar 1;13(5):1058–1064. doi: 10.1002/j.1460-2075.1994.tb06354.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fattal E., Nir S., Parente R. A., Szoka F. C., Jr Pore-forming peptides induce rapid phospholipid flip-flop in membranes. Biochemistry. 1994 May 31;33(21):6721–6731. doi: 10.1021/bi00187a044. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Gaedigk-Nitschko K., Schlesinger M. J. Site-directed mutations in Sindbis virus E2 glycoprotein's cytoplasmic domain and the 6K protein lead to similar defects in virus assembly and budding. Virology. 1991 Jul;183(1):206–214. doi: 10.1016/0042-6822(91)90133-v. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Hahn C. S., Rice C. M., Strauss E. G., Lenches E. M., Strauss J. H. Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions. J Virol. 1989 Aug;63(8):3459–3465. doi: 10.1128/jvi.63.8.3459-3465.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harrison S. C., Strong R. K., Schlesinger S., Schlesinger M. J. Crystallization of Sindbis virus and its nucleocapsid. J Mol Biol. 1992 Jul 5;226(1):277–280. doi: 10.1016/0022-2836(92)90141-6. [DOI] [PubMed] [Google Scholar]
  14. Helenius A., Kielian M., Wellsteed J., Mellman I., Rudnick G. Effects of monovalent cations on Semliki Forest virus entry into BHK-21 cells. J Biol Chem. 1985 May 10;260(9):5691–5697. [PubMed] [Google Scholar]
  15. Ivanova L., Schlesinger M. J. Site-directed mutations in the Sindbis virus E2 glycoprotein identify palmitoylation sites and affect virus budding. J Virol. 1993 May;67(5):2546–2551. doi: 10.1128/jvi.67.5.2546-2551.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Justman J., Klimjack M. R., Kielian M. Role of spike protein conformational changes in fusion of Semliki Forest virus. J Virol. 1993 Dec;67(12):7597–7607. doi: 10.1128/jvi.67.12.7597-7607.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Landsberger F. R., Compans R. W. Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus. Biochemistry. 1976 Jun 1;15(11):2356–2360. doi: 10.1021/bi00656a017. [DOI] [PubMed] [Google Scholar]
  20. Lenard J., Compans R. W. The membrane structure of lipid-containing viruses. Biochim Biophys Acta. 1974 Apr 8;344(1):51–94. doi: 10.1016/0304-4157(74)90008-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Lopez S., Yao J. S., Kuhn R. J., Strauss E. G., Strauss J. H. Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses. J Virol. 1994 Mar;68(3):1316–1323. doi: 10.1128/jvi.68.3.1316-1323.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Marquardt M. T., Phalen T., Kielian M. Cholesterol is required in the exit pathway of Semliki Forest virus. J Cell Biol. 1993 Oct;123(1):57–65. doi: 10.1083/jcb.123.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Marsh M., Helenius A. Adsorptive endocytosis of Semliki Forest virus. J Mol Biol. 1980 Sep 25;142(3):439–454. doi: 10.1016/0022-2836(80)90281-8. [DOI] [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. 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]
  29. Moore N. F., Barenholz Y., Wagner R. R. Microviscosity of togavirus membranes studied by fluorescence depolarization: influence of envelope proteins and the host cell. J Virol. 1976 Jul;19(1):126–135. doi: 10.1128/jvi.19.1.126-135.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nieva J. L., Bron R., Corver J., Wilschut J. Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane. EMBO J. 1994 Jun 15;13(12):2797–2804. doi: 10.1002/j.1460-2075.1994.tb06573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pessin J. E., Glaser M. Budding of Rous sarcoma virus and vesicular stomatitis virus from localized lipid regions in the plasma membrane of chicken embryo fibroblasts. J Biol Chem. 1980 Oct 10;255(19):9044–9050. [PubMed] [Google Scholar]
  32. Repges-Illguth S., Kaluza G. Differences in antigenicity of E2 in Semliki Forest virus particles and in infected cells. Arch Virol. 1994;135(3-4):433–435. doi: 10.1007/BF01310027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rice C. M., Strauss J. H. Nucleotide sequence of the 26S mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2062–2066. doi: 10.1073/pnas.78.4.2062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sanz M. A., Pérez L., Carrasco L. Semliki Forest virus 6K protein modifies membrane permeability after inducible expression in Escherichia coli cells. J Biol Chem. 1994 Apr 22;269(16):12106–12110. [PubMed] [Google Scholar]
  35. Schlesinger M. J., London S. D., Ryan C. An in-frame insertion into the Sindbis virus 6K gene leads to defective proteolytic processing of the virus glycoproteins, a trans-dominant negative inhibition of normal virus formation, and interference in virus shut off of host-cell protein synthesis. Virology. 1993 Mar;193(1):424–432. doi: 10.1006/viro.1993.1139. [DOI] [PubMed] [Google Scholar]
  36. Simons K., Fuller S. D. Cell surface polarity in epithelia. Annu Rev Cell Biol. 1985;1:243–288. doi: 10.1146/annurev.cb.01.110185.001331. [DOI] [PubMed] [Google Scholar]
  37. Strauss E. G., Birdwell C. R., Lenches E. M., Staples S. E., Strauss J. H. Mutants of Sindbis virus. II. Characterization of a maturation-defective mutant, ts103. Virology. 1977 Oct 1;82(1):122–149. doi: 10.1016/0042-6822(77)90038-1. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Vénien-Bryan C., Fuller S. D. The organization of the spike complex of Semliki Forest virus. J Mol Biol. 1994 Feb 18;236(2):572–583. doi: 10.1006/jmbi.1994.1166. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. 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]
  43. Zhao H., Lindqvist B., Garoff H., von Bonsdorff C. H., Liljeström P. A tyrosine-based motif in the cytoplasmic domain of the alphavirus envelope protein is essential for budding. EMBO J. 1994 Sep 15;13(18):4204–4211. doi: 10.1002/j.1460-2075.1994.tb06740.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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