Skip to main content
SpringerLink
Log in

Expression and cellular localization of the Nef protein from human immunodeficiency virus-1 in stably transfected B-cells

  • Original Papers
  • Published:
Archives of Virology Aims and scope Submit manuscript

Summary

Nef protein, encoded by the regulatorynef gene of human immunodeficiency virus type 1 (HIV-1), was expressed in the B-cell line Raji. The cells were stably transfected with plasmids containing thenef transcriptional cassette. They expressed Nef with an Mr of 27,000; the yield could be augmented by incubation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The intracellular localization of Nef was analyzed applying immunofluorescence microscopy using a confocal laser scanning microscope. The antigen was stained with a monoclonal antibody directed against the N-terminal part of Nef. The experiments revealed that in non-dividing cells Nef is present both in the cytoplasm and the nucleus while in dividing cells the viral protein is present in the cytoplasm and at the nuclear membrane.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ahmad N, Venkatesan S (1988) NEF protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 241: 1481–1485

    Google Scholar 

  2. Allan JS, Coligan JE, Lee TH, McLane MF, Kanki PJ, Groopman JE, Essex M (1985) A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients. Science 230: 810–813

    Google Scholar 

  3. Bachelerie F, Alcami J, Hazan U, Israel N, Goud B, Arenzana-Seisdedos F, Virelizier J-L (1990) Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity. J Virol 64: 3059–3062

    Google Scholar 

  4. Bachmann M, Chang SE, Slor H, Kukulies J, Müller WEG (1990) Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes. Exp Cell Res 191: 171–180

    Google Scholar 

  5. Chakrabarti L, Guyader M, Alizon M, Daniel MD, Desrosiers RC, Tiollais P, Sonigo P (1987) Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses. Nature 328: 543–547

    Google Scholar 

  6. Cheng-Mayer C, Iannello P, Shaw K, Luciw PA, Levy JA (1989) Differential effects of nef on HIV replication: implications for viral pathogenesis in the host. Science 246: 1629–1632

    Google Scholar 

  7. Chu G, Hayakawa H, Berg P (1987) Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15: 1311–1326

    Google Scholar 

  8. Clavel F, Guyader M, Guetard MS, Salle M, Montagnier L, Alizon M (1986) Molecular cloning and polymorphism of the human immune deficiency virus type 2. Nature 324: 691–695

    Google Scholar 

  9. Fisher AG, Ratner L, Mitsuya H, Marselle LM, Harper ME, Broder S, Gallo RC, Wong-Staal F (1986) Infectious mutants of HTLV-III with changes in the 3′ region and markedly reduced cytopathic effects. Science 233: 655–659

    Google Scholar 

  10. Franchini G, Robert-Guroff M, Ghrayeb J, Chang NT, Wong-Staal F (1986) Cytoplasmic localization of the HTLV-III 3′orf protein in cultured T cells. Virology 155: 593–599

    Google Scholar 

  11. Garcia JV, Miller D (1991) Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 350: 508–511

    Google Scholar 

  12. Greene WC (1991) The molecular biology of human immunodeficiency virus type 1 infection. N Engl J Med 324: 308–317

    Google Scholar 

  13. Guy B, Kieny MP, Riviere Y, Peuch, Le C, Dott K, Girad M, Montagnier L, Lecocq JP (1987) HIV F/3′ orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature 330: 266–269

    Google Scholar 

  14. Hammes SR, Dixon EP, Malim MH, Cullen BR, Greene WC (1989) Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor. Proc Natl Acad Sci USA 86: 9549–9553

    Google Scholar 

  15. Kaminchik J, Bashan N, Itach A, Sarver N, Gorecki M, Panet A (1991) Genetic characterization of human immunodeficiency virus type 1 NEF gene products translated in vitro and expressed in mammalian cells. J Virol 65: 583–588

    Google Scholar 

  16. Kienzle N, Bröker M, Harthus H-P, Enders M, Erfle V, Buck M, Müller-Lantzsch N (1991) Immunological study of the Nef protein from HIV-1 by polyclonal and monoclonal antibodies. Arch Virol 118: 29–41

    Google Scholar 

  17. Kim S, Ikeuchi K, Byrn R, Groopman J, Baltimore D (1989) Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proc Natl Acad Sci USA 86: 9544–9548

    Google Scholar 

  18. Lebkowski JS, McNally MA, Okarma TB, Lerch LB (1987) Inducible gene expression from multiple promotors by the tumor-promoting agent, PMA. Nucleic Acid Res 15: 9043–9054

    Google Scholar 

  19. Luciw PA, Cheng-Mayer C, Levy JA (1987) Mutational analysis of the human immunodeficiency virus: The orf-B region down-regulates virus replication. Proc Natl Acad Sci USA 84: 1434–1438

    Google Scholar 

  20. Müller WEG, Reuter P, Kuchino Y, Rooyen J v, Schröder HC (1989) Inhibitory effect of nonviable preparations from human immunodeficiency virus 1 on inositol phospholipid metabolism. Eur J Biochem 183: 391–396

    Google Scholar 

  21. Niederman TMJ, Thielan BJ, Ratner L (1989) Human immunodeficiency virus type 1 negative factor is a transcriptional silencer. Proc Natl Acad Sci USA 86: 1128–1132

    Google Scholar 

  22. Ovod V, Lagerstedt A, Ranki A, Gombert F, Schaude R, Tähtinen M, Jung G, Krohn K (1991) Phenotypic variation in HIV-1 Nef demonstrated with a panel of eptitope specific monoclonal antibodies. In: VII International Conference on AIDS, Florence, 16–21 June, 1991, abstract TU.A. 73

  23. Pulvertaft RJV (1965) A study of malignant tumors in Nigeria by short-term tissue culture. J Clin Pathol 18: 261–271

    Google Scholar 

  24. Samuel KP, Seth A, Konopka A, Lautenberger JA, Papas TS (1987) The 3′-orf protein of human immunodeficiency virus shows structural homology with the phosphorylation domain of human interleukin-2 receptor and the ATP-binding site of the protein kinase family. FEBS Lett 218: 81–86

    Google Scholar 

  25. Stinski MF, Roehr TJ (1985) Activation of the major immediate early gene of human cytomegalovirus by cis-acting elements in the promotor-regulatory sequence and by virus-specific trans acting components. J Virol 55: 431–441

    Google Scholar 

  26. Terwilliger E, Sodrosky J, Rosen CA, Haseltine WA (1986) Effects of mutations within the 3′ orf open reading frame region of human T-cell lymphotropic virus type III (HTLVIII/LAV) on replication and cytopathogenicity. J Virol 60: 754–760

    Google Scholar 

  27. Vega MA, Guigo R, Smith TF (1990) Autoimmune response in AIDS. Nature 345: 26

    Google Scholar 

  28. Wain-Hobson S, Sonigo P, Danos O, Cole S, Alizon M (1985) Nucleotide sequence of the AIDS virus, LAV. Cell 40: 9–17

    Google Scholar 

  29. Weinshenker BG, Wilton S, Rice GP (1988) Phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line. J Immunol 140: 1625–1631

    Google Scholar 

  30. Wolber V, Rensland H, Brandmeier B, Wittinghofer F, Kalbitzer HR (1991) Does the HIV protein bind guanine nucleotides? In: VII International Conference on AIDS, Florence, 16–21 June, 1991, abstract M.A. 1204

  31. Yates JL, Warren N, Sugden B (1985) Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313: 812–815

    Google Scholar 

  32. Young JM, Cheadle C, Foulke JS Jr, Drohan WN, Sarver N (1988) Utilization of an Epstein-Barr virus replicon as a eukaryotic expression vector. Gene 62: 171–185

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Homburg, Homburg/Saar, Federal Republic of Germany

    N. Kienzle & N. Müller-Lantzsch

  2. Abteilung Angewandte Molekularbiologie, Institut für Physiologische Chemie, Universität Mainz, Mainz, Federal Republic of Germany

    M. Bachmann & W. E. G. Müller

Authors
  1. N. Kienzle
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Bachmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. W. E. G. Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N. Müller-Lantzsch
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kienzle, N., Bachmann, M., Müller, W.E.G. et al. Expression and cellular localization of the Nef protein from human immunodeficiency virus-1 in stably transfected B-cells. Archives of Virology 124, 123–132 (1992). https://doi.org/10.1007/BF01314630

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01314630

Keywords

Search

Navigation