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Immunological study of the nef protein from HIV-1 by polyclonal and monoclonal antibodies

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Summary

We constructed and expressed different overlapping fusion proteins with the nef gene of HIV-1 and generated specific polyclonal rabbit and monoclonal mouse antibodies against these recombinant proteins. The rabbit antisera, one of the monoclonal antibodies as well as a serum from a HIV-1 infected patient recognized the nef protein with Mr 27 kDa in latently HIV-1 infected glioma cells in the immunoblot. In contrast, these antibodies could not detect nef in productively HIV-1 infected Molt-3 cells neither in immunoblot nor in indirect immunofluorescence assays. These results indicate the possible participation of nef in viral latency.

The recombinant nef proteins were used as probes for anti-nef antibodies in human sera. We observed in 17 of 57 sera tested specific anti-nef antibodies. All of these anti-nef positive sera also contained antibodies directed against viral structural proteins. The NH2-terminal region of the recombinant nef was shown to be the major immunodominant antigenic site in the immunoblot assay.

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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, 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. Ameisen J-C, Guy B, Chamaret S, Loche M, Mouton Y, Neyrinck J-L, Khalife J, Leprevost C, Beaucaire G, Boutillon C, Gras-Masse H, Maniez M, Kieny M-P, Laustriat D, Berthier A, Mach B, Montagnier L, Lecocq J-P, Capron A (1989) Antibodies to the nef protein and to nef peptides in HIV-1-infected seronegative individuals. Aids Res Human Retrovir 5: 279–291

    Google Scholar 

  4. Appleyard RK (1954) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived fromEscherichia coli K12. Genetics 39: 440–452

    Google Scholar 

  5. Arya SK (1987) 3′-orf and sor genes of human immunodeficiency virus: in vitro transcription-translation and immunoreactive domains. Proc Natl Acad Sci USA 84: 5429–5433

    Google Scholar 

  6. Arya SK, Gallo RC (1986) Three novel genes of human T-lymphotropic virus type III: immune reactivity of their products with sera from acquired immune deficiency syndrome patients. Proc Natl Acad Sci USA 83: 2209–2213

    Google Scholar 

  7. Bröker M (1986) Vectors for regulated high-level expression of proteins fused to truncated forms ofEscherichia coli β-galactosidase. Gene Anal Techn 3: 53–57

    Google Scholar 

  8. Burnette N (1981) “Western Blotting”: electrophoretic transfer of protein from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195–203

    Google Scholar 

  9. 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 

  10. 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 

  11. 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 

  12. Cohen EA, Terwilliger EF, Sodroski JG, Haseltine WA (1988) Identification of a protein encoded by the vpu gene of HIV-1. Nature 334: 532–534

    Google Scholar 

  13. 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 

  14. 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 

  15. Gallo RC, Wong-Staal F, Montagnier L, Haseltine W, Yoshida M (1988) HIV/HTLV gene nomenclature. Nature 333: 504

    Google Scholar 

  16. Gombert FO, Blecha W, Tähtinen M, Ranki A, Pfeifer S, Tröger W, Braun R, Müller-Lantzsch N, Jung G, Rübsamen-Waigmann H, Krohn K (1990) Antigenic epitopes of NEF proteins from different HIV-1 strains as recognized by sera from patients with manifest and latent infection. Virology 176: 458–466

    Google Scholar 

  17. Guy B, Kieny MP, Riviere Y, Le Peuch 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 

  18. 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 

  19. Harlow E, Lane D (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  20. Haseltine WA (1989) Silent HIV infections. New Engl J Med 320: 1487–1489

    Google Scholar 

  21. 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 

  22. Koenen M, Rüther U, Müller-Hill B (1982) Immunoenzymatic detection of expressed gene fragments cloned in the LacZ gene ofE. coli. EMBO J 1: 509–512

    Google Scholar 

  23. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685

    Google Scholar 

  24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275

    Google Scholar 

  25. 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 

  26. Mellert W, Festl H, Kleinschmidt A, Müller-Lantzsch N, Erfle V (1989) Cell-specific control of HIV production in chronically infected glial cells. In: Fifth international conference on AIDS, abstract no W.C.P.91

  27. Müller-Lantzsch N, Fan H (1976) Monospecific immunoprecipitation of murine leukemia virus polyribosomes: identification of p30 protein-specific messenger RNA. Cell 9: 579–588

    Google Scholar 

  28. Müller-Lantzsch N, Lenoir GM, Sauter M, Takaki K, Bechet J-M, Kuklik-Ross C, Wunderlich D, Bornkamm GW (1985) Identification of the coding region for a second Epstein-Barr virus nuclear antigen (EBNA-2) by transfection of cloned DNA fragments. EMBO J 4: 1805–1811

    Google Scholar 

  29. 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 

  30. Popovic M, Read-Connole E, Gallo RC (1984) T4 positive human neoplastic cell lines susceptible to and permissive for HTLV-III. Lancet ii: 1472–1473

    Google Scholar 

  31. Ranki A, Krohn M, Allain JP, Franchini G, Valle SL, Antonen J, Leuther M, Krohn K (1987) Long latency precedes overt seroconversion in sexually transmitted human-immunodeficiency-virus infection. Lancet ii: 589–593

    Google Scholar 

  32. Ratner L, Haseltine W, Patarca R, Livak KJ, Starcich B, Josephs SF, Doran ER, Rafalski Ja, Whitehozn EA, Baumeister K, Ivanoff L, Petteway SR, Pearson ML, Lautenberger JA, Papas TS, Ghrayeb J, Chang NT, Gallo RC, Wong-Staal F (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313: 277–284

    Google Scholar 

  33. Ratner L, Starcich B, Josephs SF, Hahn BH, Reddy EP, Livak KJ, Petteway SR, Pearson ML, Haseltine WA, Arya SK, Wong-Staal F (1985) Polymorphism of the 3′ open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III. Nucleic Acids Res 13: 8219–8229

    Google Scholar 

  34. Reiss P, de Ronde A, Lange JMA, de Wolf F, Dekker J, Debouck C, Goudsmit J (1989) Antibody response to the viral negative factor (nef) in HIV-1 infection: a correlate of levels of HIV-1 expression. AIDS 3: 227–233

    Google Scholar 

  35. Riviere Y, Tanneau-Salvadori F, Regnault A, Lopez O, Sansonetti P, Guy B, Kieny M-P, Fournel J-J, Montagnier L (1989) Human immunodeficiency virus-specific cytotoxic response of seropositive individuals: distinct types of effector cells mediate killing of targets expressing gag and env proteins. J Virol 63: 2270–2277

    Google Scholar 

  36. Sabatier J-M, Clerget-Raslain B, Fontan G, Fenouillet E, Rochat H, Granier C, Gluckman J-C, Rietschoten JV, Montagnier L, Bahraoui (1989) Use of synthetic peptides for the detection of antibodies against the nef regulating protein in sera of HIV-infected patients. AIDS 3: 215–220

    Google Scholar 

  37. Samuel KP, Seth A, Konopka A, Lautenberger JA, Papas Ts (1987) The 3′ orf protein of human immunodeficiency virus shows structural homolgy 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 

  38. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467

    Google Scholar 

  39. Sauter M, Müller-Lantzsch N (1987) Characterization of an Epstein-Barr virus nuclear antigen 2 variant (EBNA 2B) by specific sera. Virus Res 8: 141–152

    Google Scholar 

  40. Spindler K, Rosser D, Berk A (1984) Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced inEscherichia coli. J Virol 49: 132–141

    Google Scholar 

  41. 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 

  42. 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 

  43. Walter G, Scheidtmann K-H, Carbone A, Laudono AP, Doolitle RF (1980) Antibodies specific for the carboxy- and aminoterminal regions of simian virus 40 large tumor antigen. Proc Natl Acad Sci USA 77: 5197–5200

    Google Scholar 

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Authors and Affiliations

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

    N. Kienzle, M. Enders, M. Buck & N. Müller-Lantzsch

  2. Behringwerke AG, Marburg

    M. Bröker & H. -P. Harthus

  3. GSF-Abteilung für Molekulare Zellpathologie, Neuherberg, Federal Republic of Germany

    V. Erfle

Authors
  1. N. Kienzle
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  2. M. Bröker
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  3. H. -P. Harthus
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  4. M. Enders
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  5. V. Erfle
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  6. M. Buck
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  7. N. Müller-Lantzsch
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Kienzle, N., Bröker, M., Harthus, H.P. et al. Immunological study of the nef protein from HIV-1 by polyclonal and monoclonal antibodies. Archives of Virology 118, 29–41 (1991). https://doi.org/10.1007/BF01311301

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  • DOI: https://doi.org/10.1007/BF01311301

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