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Volume 78, Issue 2

Regulated expression vectors demonstrate cell-type-specific sensitivity to human immunodeficiency virus type 1 Nef-induced cytostasis Free

Abstract

The gene product of both human and simian immunodeficiency viruses is critically important for virus replication and disease progression . However, the precise biological function of Nef remains poorly characterized , with previous reports suggesting that Nef might be either cytotoxic or cytostatic. As a result of difficulties encountered by several groups in establishing cell lines constitutively expressing Nef, we have developed two inducible systems resulting in stable Nef expression in various mammalian cell lines. Tetracycline-regulated Nef expression was achieved in HeLa cells but could not be established in human T cell lines. Jurkat E6-1 T cell and RAW264.7 murine macrophage cell lines expressing a regulated gene were generated using a system in which Nef expression was controlled by a mutated version of the heavy metal-inducible human metallothionein IIA promoter. Induction of high levels of Nef expression in HeLa-Nef and Jurkat-Nef cells resulted in a moderate (2-fold) and a dramatic (10-fold) retardation of cell growth respectively, supporting the contention that Nef may be a cytotoxic or cytostatic factor. This property was also observed at low basal levels of Nef expression in RAW264.7-Nef macrophage clones (5-fold reduction in growth) and was associated with an altered morphological phenotype suggesting that different cell types may be more susceptible to the cytostatic activity of Nef. The regulated Nef-expression systems provide tools for investigating the molecular basis of Nef function, including Nef-mediated cytopathogenicity, CD4 down-regulation and enhancement of virus infectivity.

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© Society for General Microbiology Ltd 1997
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1997-02-01
2024-04-20
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References

  1. Ackland-Berglund C. E., Leib D. A. 1995; Efficacy of tetracycline-controlled gene expression is influenced by cell type. BioTechniques 18:196–200
     [Google Scholar]
  2. Ahmad N., Venkatesan S. 1988; Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 241:1481–1485
     [Google Scholar]
  3. Aiken C., Trono D. 1995; Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. Journal of Virology 69:5048–5056
     [Google Scholar]
  4. Aiken C., Konner J., Landau N. R., Lenburg M. E., Trono D. 1994; Nefinduces CD4 endocytosis:requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 76:853–864
     [Google Scholar]
  5. Aldrovandi G. M., Zack J. A. 1996; Replication andpathogenicity of human immunodeficiency virus type 1 accessory gene mutants in SCID- Hu mice. Journal of Virology 70:1505–1511
     [Google Scholar]
  6. Allan J. S., Coligan J. E., Lee T., McLane M. F., Kanki P. J., Groopman J. E., Essex M. 1985; A new HTLV-III/LAV encoded antigen detectedby antibodies fromAIDS patients. Science 230:810–813
     [Google Scholar]
  7. Anderson S., Shugars D. C., Swanstrom R., Garcia J. V. 1993; Nef from primary isolates ofhuman immunodeficiency virus type 1 suppresses surface CD4 expression in human and mouse T cells. Journal of Virology 67:4923–4931
     [Google Scholar]
  8. Bandres J. C., Ratner L. 1994; Human immunodeficiency virus type 1 Nef protein down-regulates transcription factors NFKB and AP-1 in human T cells in vitro after T-cell receptor stimulation. Journal of Virology 68:3243–3249
     [Google Scholar]
  9. Baur A. S., Sawai E. T., Dazin P., Fantl W. J., Cheng-Mayer C., Peterlin B. M. 1994; HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization. Immunity 1:373–384
     [Google Scholar]
  10. Benson R. E., Sanfridson A., Ottinger J. S., Doyle C., Cullen B. R. 1993; Downregulation of cell-surface CD4 expression by simian immunodeficiency virus nef prevents viral super infection. Journal of Experimental Medicine 177:1561–1566
     [Google Scholar]
  11. Brady H. J. M., Pennington D. J., Miles C. G., Dzierzak E. A. 1993; CD4 cell surface downregulation in HIV-1 nef transgenic mice is a consequence of intracellular sequestration. EMBO Journal 12:4923–4932
     [Google Scholar]
  12. Chowers M. Y., Spina C. A., Kwoh T. J., Fitch N. J. S., Richman D. D., Guatelli J. C. 1994; Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene. Journal of Virology 68:2906–2914
     [Google Scholar]
  13. Collette Y., Chang H. L., Cerdan C., Chambost H., Algarte M., Mawas C., Imbert J., Burny A., Olive D. 1996a; Specific Th1 cytokine down-regulation associated with primary clinically derived human immunodeficiency virus type 1 nef gene-induced expression. Journal of Immunology 156:360–370
     [Google Scholar]
  14. Collette Y., Dutartre H., Benziane A., Ramosmorales F., Benarous R., Harris M., Olive D. 1996b; Physical and functional interaction of Nef with lck - HIV-1 Nef-induced T-cell signalling defects. Journal of Biological Chemistry 271:6333–6341
     [Google Scholar]
  15. Clapham P. R., McKnight A., Weiss R. A. 1992; Human immunodeficiency virus type 2 infection and fusion of CD4-negative cell lines: induction and enhancement by soluble CD4. Journal of Virology 66:3531–3537
     [Google Scholar]
  16. Clavel F., Charneau P. 1994; Fusion from without directed by human immunodeficiency virus particles. Journal of Virology 68:1179–1185
     [Google Scholar]
  17. Deacon N. J., Tsykin A., Solomon A., Smith K., Ludford-Menting M., Hooker D. J., McPhee D. A., Greenway A. L., Ellett A., Chatfield C., Lawson V. A., Crowe S., Maerz A., Sonza S., Learmont J., Sullivan J. S., Cunningham A., Dwyer D., Dowton D., Mills J. 1995; Genomic structure of an attenuated quasi-species of HIV-1 from a blood transfusion donor and recipients. Science 270:988–991
     [Google Scholar]
  18. Du Z., Lang S. M., Sasseville V. G., Lackner A. A., Ilyinskil P. O., Daniel M. D., Jung J. U., Desrosiers R. C. 1995; Identification of a nef allele that causes lymphocyte activation and acute disease in macaque monkeys. Cell 82:665–674
     [Google Scholar]
  19. Garcia J. V., Miller A. D. 1991; Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 350:508–511
     [Google Scholar]
  20. Garcia J. V., Alfano J., Miller A. D. 1993; The negative effect of human immunodeficiency virus type 1 Nef on cell surface CD4 expression is not species specific and requires the cytoplasmic domain of CD4. Journal of Virology 67:1511–1516
     [Google Scholar]
  21. Gossen M., Bujard H. 1992; Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences USA: 895547–5551
     [Google Scholar]
  22. Greenway A., Azad A., McPhee D. 1995; Human immunodeficiency virus type 1 Nef protein inhibits activation pathways in peripheral blood mononuclear cells and T-cell lines. Journal of Virology 69:1842–1850
     [Google Scholar]
  23. Guy B., Kieny M. P., Riviere Y., le Peuch C., Dott K., Girard M., Montagnier L., Lecocq J. 1987; HIV F/3′ orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature 330:266–269
     [Google Scholar]
  24. Hammes S. R., Dixon E. P., Malim M. H., Cullen B. R., Greene W. C. 1989; Nef protein of human immunodeficiency virus type 1 : evidence against its role as a transcriptional inhibitor. Proceedings of the National Academy of Sciences USA: 869549–9553
     [Google Scholar]
  25. Harris M. P. G., Neil J. C. 1994; Myristoylation-dependent binding of HIV-1 Nef to CD4. Journal of Molecular Biology 241:136–142
     [Google Scholar]
  26. Harris M., Hislop S., Patsilinacos P., Neil J. C. 1992; In vivoderived HIV-1 nef gene products are heterogeneous and lack detectable nucleotide binding activity. AIDS Research and Human Retroviruses 8:537–543
     [Google Scholar]
  27. Jamieson B. D., Aldrovandi G. M., Planelles V., Jowett J. B. M., Gao L., Bloch L. M., Chen I. S. Y., Zack J. A. 1994; Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity. Journal of Virology 68:3478–3485
     [Google Scholar]
  28. Kestler H. W., Ringler D. J., Mori K., Panicali D. L., Sehgal P. K., Daniel M. D., Desrosiers R. C. 1991; Importance ofthe nef gene for maintenance of high virus loads and for development of AIDS. Cell 65:651–662
     [Google Scholar]
  29. 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. Proceedings of the National Academy of Sciences USA: 869544–9548
     [Google Scholar]
  30. Luciw P. A., Cheng-Mayer C., Levy J. A. 1987; Mutational analysis of the human immunodeficiency virus: the orf-B region down-regulates virus replication. Proceedings of the National Academy of Sciences USA: 841434–1438
     [Google Scholar]
  31. Luria S., Chambers I., Berg P. 1991; Expression of the type 1 human immunodeficiency virus Nef protein in T cells prevents antigen receptor-mediated induction of interleukin 2 mRNA. Proceedings of the National Academy of Sciences USA: 885326–5330
     [Google Scholar]
  32. McKeating J. A., Bennett J., Zolla-Pazner S., Schutten M., Ashelford S., Leigh Brown A. J., Balfe P. 1993; Resistance of a human serum selected human immunodeficiency virus type 1 escape mutant to neutralization by CD4 binding site monoclonal antibodies is conferred by a single amino acid change in gp120. Journal of Virology 67:5216–5225
     [Google Scholar]
  33. Makarov S. S., Jonat C., Haskill S. 1994; Hyperinducible human metallothionein promoter with a low level basal activity. Nucleic Acids Research 22:1504–1505
     [Google Scholar]
  34. Mariani R., Skowronski J. 1993; CD4 down-regulation by nef alleles isolated from human immunodeficiency virus type 1-infected individuals. Proceedings of the National Academy of Sciences USA: 905549–5553
     [Google Scholar]
  35. Miller M. D., Warmerdam M. T., Gaston I., Greene W. C., Feinberg M. B. 1994; The human immunodeficiency virus-1 nef gene product : a positive factor for viral infection and replication in primary lymphocytes and macrophages. Journal of Experimental Medicine 179:101–113
     [Google Scholar]
  36. Miller M. D., Warmerdam M. T., Page K. A., Feinberg M. B., Greene W. C. 1995; Expression of the human immunodeficiency virus type 1 (HIV-1) nef gene during HIV-1 production increases progeny particle infectivity independently of gp160 or viral entry. Journal of Virology 69:579–584
     [Google Scholar]
  37. Mosmann T. 1983; Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65:55–63
     [Google Scholar]
  38. Murphy K. M., Sweet M. T., Ross I. L., Hume D. A. 1993; Effects of the tat and nef gene products of human immunodeficiency virus type 1 (HIV-1) on transcription controlled by the HIV-1 long terminal repeat and on cell growth in macrophages. Journal of Virology 67:6956–6964
     [Google Scholar]
  39. Myers G., Korber B., Hahn B. H., Jeang K. T., Mellors J. W., McCutchan F. E., Henderson L. E., Pavlakis G. N. 1995 editor Human Retroviruses and AIDS 1995 Los Alamos, N. Mex: Los Alamos National Laboratory;
     [Google Scholar]
  40. Niederman T. M. J., Thielan B. J., Ratner L. 1989; Human immunodeficiency virus type I negative factor is a transcriptional silencer. Proceedings of the National Academy of Sciences USA: 861128–1132
     [Google Scholar]
  41. Niederman T. M., Garcia J. V., Hastings W. R., Luria S., Ratner L. 1992; Human immunodeficiency virus type 1 Nef protein inhibits NF- kappa B induction in human T cells. Journal of Virology 66:6213–6219
     [Google Scholar]
  42. Niederman T. M. J., Hastings W. R., Luria S., Bandres J. C., Ratner L. 1993; HIV-1 Nef protein inhibits the recruitment of AP-1 DNA- binding activity in human T-cells. Virology 194:338–344
     [Google Scholar]
  43. Raschke W., Bairds C., Ralph R., Nakoinz I. 1978; Functional macrophage cell lines transformed by Abelson leukaemia virus. Cell 15:261–267
     [Google Scholar]
  44. Rhee S. S., Marsh J. W. 1994a; HIV-1 Nef activity in murine T cells: CD4 modulation and positive enhancement. Journal of Immunology 152:5128–5134
     [Google Scholar]
  45. Rhee S. S., Marsh J. W. 1994b; Human immunodeficiency virus type 1 Nef-induced down-modulation of CD4 is due to rapid internalization and degradation of surface CD4. Journal of Virology 68:5156–5163
     [Google Scholar]
  46. Ryan-Graham M. A., Peden K. W. C. 1995; Both virus and host components are important for the manifestation of a nef- phenotype in HIV-1 and HIV-2. Virology 213:158–168
     [Google Scholar]
  47. Saksela K., Cheng G., Baltimore D. 1995; Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef + viruses but not for down- regulation of CD4. EMBO Journal 14:484–491
     [Google Scholar]
  48. Schwartz O., Dautry-Varsat A., Goud B., Marechal V., Subtil A., Heard J., Danos O. 1995a; Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes. Journal of Virology 69:528–533
     [Google Scholar]
  49. Schwartz O., Marechal V., Danos O., Heard J. M. 1995b; Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell. Journal of Virology 69:4053–4059
     [Google Scholar]
  50. Shugars D. C., Smith M. S., Glueck D. H., Nantermet P. V., Seillier-Moiseiwitsch F., Swanstrom R. 1993; Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo. Journal of Virology 67:4639–4650
     [Google Scholar]
  51. Skowronski J., Mariani R. 1995; Transient assay for Nef-induced down-regulation of CD4 antigen expression on the cell surface. In HIV: A Practical Approach pp 231–242 Karn J. Edited by Oxford: Oxford University Press;
     [Google Scholar]
  52. Skowronski J., Parks D., Mariani R. 1993; Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO Journal 12:703–713
     [Google Scholar]
  53. Spina C. A., Kwoh T. J., Chowers M. Y., Guatelli J. C., Richman D. D. 1994; The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. Journal of Experimental Medicine 179:115–123
     [Google Scholar]
  54. Terwilliger E., Sodroski J. G., Rosen C. A., Haseltine W. A. 1986; Effects of mutations within the 3′ orf open reading frame region of human T-cell lymphotropic virus type III (HTLV-III/LAV) on replication and cytopathogenicity. Journal of Virology 60:754–760
     [Google Scholar]
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Regulated expression vectors demonstrate cell-type-specific sensitivity to human immunodeficiency virus type 1 Nef-induced cytostasis
J. Gen. Virol. 78, 381 (1997); https://doi.org/10.1099/0022-1317-78-2-381
/content/journal/jgv/10.1099/0022-1317-78-2-381
/content/journal/jgv/10.1099/0022-1317-78-2-381
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