Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1997 Mar;71(3):2383–2389. doi: 10.1128/jvi.71.3.2383-2389.1997

Mutational analysis of the oligomer assembly domain in the transmembrane subunit of the Rous sarcoma virus glycoprotein.

D A Einfeld 1, E Hunter 1
PMCID: PMC191348  PMID: 9032375

Abstract

The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Bernstein H. B., Tucker S. P., Kar S. R., McPherson S. A., McPherson D. T., Dubay J. W., Lebowitz J., Compans R. W., Hunter E. Oligomerization of the hydrophobic heptad repeat of gp41. J Virol. 1995 May;69(5):2745–2750. doi: 10.1128/jvi.69.5.2745-2750.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blacklow S. C., Lu M., Kim P. S. A trimeric subdomain of the simian immunodeficiency virus envelope glycoprotein. Biochemistry. 1995 Nov 21;34(46):14955–14962. doi: 10.1021/bi00046a001. [DOI] [PubMed] [Google Scholar]
  3. Bullough P. A., Hughson F. M., Skehel J. J., Wiley D. C. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature. 1994 Sep 1;371(6492):37–43. doi: 10.1038/371037a0. [DOI] [PubMed] [Google Scholar]
  4. Carr C. M., Kim P. S. A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell. 1993 May 21;73(4):823–832. doi: 10.1016/0092-8674(93)90260-w. [DOI] [PubMed] [Google Scholar]
  5. Chen S. S. Functional role of the zipper motif region of human immunodeficiency virus type 1 transmembrane protein gp41. J Virol. 1994 Mar;68(3):2002–2010. doi: 10.1128/jvi.68.3.2002-2010.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen S. S., Lee C. N., Lee W. R., McIntosh K., Lee T. H. Mutational analysis of the leucine zipper-like motif of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein. J Virol. 1993 Jun;67(6):3615–3619. doi: 10.1128/jvi.67.6.3615-3619.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis G. L., Hunter E. A charged amino acid substitution within the transmembrane anchor of the Rous sarcoma virus envelope glycoprotein affects surface expression but not intracellular transport. J Cell Biol. 1987 Sep;105(3):1191–1203. doi: 10.1083/jcb.105.3.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Decroly E., Cornet B., Martin I., Ruysschaert J. M., Vandenbranden M. Secondary structure of gp160 and gp120 envelope glycoproteins of human immunodeficiency virus type 1: a Fourier transform infrared spectroscopic study. J Virol. 1993 Jun;67(6):3552–3560. doi: 10.1128/jvi.67.6.3552-3560.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Delwart E. L., Mosialos G., Gilmore T. Retroviral envelope glycoproteins contain a "leucine zipper"-like repeat. AIDS Res Hum Retroviruses. 1990 Jun;6(6):703–706. doi: 10.1089/aid.1990.6.703. [DOI] [PubMed] [Google Scholar]
  10. Dubay J. W., Roberts S. J., Brody B., Hunter E. Mutations in the leucine zipper of the human immunodeficiency virus type 1 transmembrane glycoprotein affect fusion and infectivity. J Virol. 1992 Aug;66(8):4748–4756. doi: 10.1128/jvi.66.8.4748-4756.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Einfeld D. A., Hunter E. Expression of the TM protein of Rous sarcoma virus in the absence of SU shows that this domain is capable of oligomerization and intracellular transport. J Virol. 1994 Apr;68(4):2513–2520. doi: 10.1128/jvi.68.4.2513-2520.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Einfeld D., Hunter E. Oligomeric structure of a prototype retrovirus glycoprotein. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8688–8692. doi: 10.1073/pnas.85.22.8688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fass D., Harrison S. C., Kim P. S. Retrovirus envelope domain at 1.7 angstrom resolution. Nat Struct Biol. 1996 May;3(5):465–469. doi: 10.1038/nsb0596-465. [DOI] [PubMed] [Google Scholar]
  14. Gallaher W. R., Ball J. M., Garry R. F., Griffin M. C., Montelaro R. C. A general model for the transmembrane proteins of HIV and other retroviruses. AIDS Res Hum Retroviruses. 1989 Aug;5(4):431–440. doi: 10.1089/aid.1989.5.431. [DOI] [PubMed] [Google Scholar]
  15. Hunter E., Hill E., Hardwick M., Bhown A., Schwartz D. E., Tizard R. Complete sequence of the Rous sarcoma virus env gene: identification of structural and functional regions of its product. J Virol. 1983 Jun;46(3):920–936. doi: 10.1128/jvi.46.3.920-936.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hunter E., Swanstrom R. Retrovirus envelope glycoproteins. Curr Top Microbiol Immunol. 1990;157:187–253. doi: 10.1007/978-3-642-75218-6_7. [DOI] [PubMed] [Google Scholar]
  17. Katz R. A., Omer C. A., Weis J. H., Mitsialis S. A., Faras A. J., Guntaka R. V. Restriction endonuclease and nucleotide sequence analyses of molecularly cloned unintegrated avian tumor virus DNA: structure of large terminal repeats in circle junctions. J Virol. 1982 Apr;42(1):346–351. doi: 10.1128/jvi.42.1.346-351.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lu M., Blacklow S. C., Kim P. S. A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol. 1995 Dec;2(12):1075–1082. doi: 10.1038/nsb1295-1075. [DOI] [PubMed] [Google Scholar]
  19. Patarca R., Haseltine W. A. Similarities among retrovirus proteins. Nature. 1984 Dec 6;312(5994):496–496. doi: 10.1038/312496a0. [DOI] [PubMed] [Google Scholar]
  20. Perez L. G., Davis G. L., Hunter E. Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions. J Virol. 1987 Oct;61(10):2981–2988. doi: 10.1128/jvi.61.10.2981-2988.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pinter A., Honnen W. J., Tilley S. A., Bona C., Zaghouani H., Gorny M. K., Zolla-Pazner S. Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1. J Virol. 1989 Jun;63(6):2674–2679. doi: 10.1128/jvi.63.6.2674-2679.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rey M. A., Krust B., Laurent A. G., Montagnier L., Hovanessian A. G. Characterization of human immunodeficiency virus type 2 envelope glycoproteins: dimerization of the glycoprotein precursor during processing. J Virol. 1989 Feb;63(2):647–658. doi: 10.1128/jvi.63.2.647-658.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schulz T. F., Jameson B. A., Lopalco L., Siccardi A. G., Weiss R. A., Moore J. P. Conserved structural features in the interaction between retroviral surface and transmembrane glycoproteins? AIDS Res Hum Retroviruses. 1992 Sep;8(9):1571–1580. doi: 10.1089/aid.1992.8.1571. [DOI] [PubMed] [Google Scholar]
  24. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  25. Tucker S. P., Srinivas R. V., Compans R. W. Molecular domains involved in oligomerization of the Friend murine leukemia virus envelope glycoprotein. Virology. 1991 Dec;185(2):710–720. doi: 10.1016/0042-6822(91)90542-j. [DOI] [PubMed] [Google Scholar]
  26. White J. M. Membrane fusion. Science. 1992 Nov 6;258(5084):917–924. doi: 10.1126/science.1439803. [DOI] [PubMed] [Google Scholar]
  27. Wild C., Dubay J. W., Greenwell T., Baird T., Jr, Oas T. G., McDanal C., Hunter E., Matthews T. Propensity for a leucine zipper-like domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12676–12680. doi: 10.1073/pnas.91.26.12676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wild C., Oas T., McDanal C., Bolognesi D., Matthews T. A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10537–10541. doi: 10.1073/pnas.89.21.10537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wiley D. C., Skehel J. J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem. 1987;56:365–394. doi: 10.1146/annurev.bi.56.070187.002053. [DOI] [PubMed] [Google Scholar]
  30. Wills J. W., Srinivas R. V., Hunter E. Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products. J Cell Biol. 1984 Dec;99(6):2011–2023. doi: 10.1083/jcb.99.6.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES