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Purification of RBF-2, a transcription factor with specificity for the most conservedcis-element of naturally occurring HIV-1 LTRs

  • Original Paper
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Journal of Biomedical Science

Abstract

The combination of high turnover and error-prone reverse transcription results in naturally occurring human immunodeficiency virus-1 long terminal repeats that differ considerably from the prototype sequence. Although no transcription-factor-binding site escapes mutation, the only mutated site that appears to be invariably compensated by co-occurrence of its duplication is the RBE III site, a target for the transcription factor RBF-2. In this work, we characterize RBF-2 further by biochemical purification. RBF-2 was purified by chromatography on heparin agarose and Mono-Q ion exchange chromatography, followed by affinity chromatography on mutant and wild-type RBE III oligonucleotide columns. The purified RBF-2 preparation contained 4 major and 1 minor polypeptides of 50, 100, 110, 120 and 125 kD, as detected by silver staining in SDS-PAGE gels. UV cross-linking revealed a specific 100-kD species, indicating that this protein likely represents the DNA-binding component of a complex. A second factor with DNA-binding specificity similar to that of RBF-2, called RBF-B, was also identified by heparin-agarose fractionation, which suggests that effects of the RBE IIIcis-element may be mediated by a combination of factors in vivo.

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References

  1. Ait-Khaled M, Emery VC. Phylogenetic relationship between human immunodeficiency virus type 1 (HIV-1) long terminal repeat natural variants present in the lymph node and peripheral blood of three HIV-1-infected individuals. J Gen Virol 75:1615–21;1994.

    Google Scholar 

  2. Ait-Khaled M, McLaughlin JE, Johnson MA, Emery VV. Distinct HIV-1 long terminal repeat quasispecies present in nervous tissues compared to that in lung, blood and lymphoid tissues of an AIDS patient. AIDS 9:675–683;1995.

    Google Scholar 

  3. Alizon M, Wain-Hobson S, Montagnier L, Sonigo P. Genetic variability of the AIDS virus: Nucleotide sequence analysis of two isolates from African patients. Cell 46:63–74;1986.

    Google Scholar 

  4. Bell B, Sadowski I. Ras-responsiveness of the HIV-1 LTR requires RBF-1 and RBF-2 binding sites. Oncogene 13:2687–2697;1996.

    Google Scholar 

  5. Bowdish KS, Yuan HE, Mitchel AP. Positive control of yeast meiotic genes by the negative regulator UME6. Mol Cell Biol 15:2955–2961;1995.

    Google Scholar 

  6. Cheynier R, Henichwark S, Hadida F, Pelletier E, Oksenhendler E, Autran B, Wain-Hobson S. HIV and T cell expansion in splenic white pulps is accompanied by infiltration of HIV-specific cytotoxic T lymphocytes. Cell 78:373–387;1994.

    Google Scholar 

  7. Cheynier R, Henrichwark S, Hadida F, Pelletier E, Oksenhendler E, Autran B, Wain-Hobson S. Clonal expansion of T cells and HIV genotypes in microdissected splenic white pulps indicates viral replication in situ and infiltration of HIV-specific cytotoxic T lymphocytes. Adv Exp Med Biol 374:173–182;1995.

    Google Scholar 

  8. Coffin JM. HIV population dynamics in vivo: Implications for genetic variation, pathogenesis and therapy. Science 267:483–489;1995.

    Google Scholar 

  9. Delassus S, Meyerhans A, Cheynier R, Wain-Hobson S. Absence of selection of HIV-1 variants in vivo based on transcription/transactivation during progression to AIDS. Virology 188:811–818;1992.

    Google Scholar 

  10. Estable MC, Bell B, Hirst M, Sadowski IJ. Naturally occurring HIV-1 LTRs have a frequently observed duplication that binds RBF-2 and represses transcription. J Virol 72:6465–6474;1998.

    Google Scholar 

  11. Estable MC, Bell B, Merzouki A, Montaner JS, O'Shaughnessy MV, Sadowski IJ. Human immunodeficiency virus type 1 long terminal repeat variants from 42 patients representing all stages of infection display a wide range of sequence polymorphism and transcription activity. J Virol 70:4053–4062;1996.

    Google Scholar 

  12. Estable MC, Merzouki A, Arella M, Sadowski IJ. Distinct clustering of HIV-1 sequences derived from injection drug users versus non-injection drug in users in Vancouver, Canada. AIDS Res Hum Retroviruses 14:917–919;1998.

    Google Scholar 

  13. Fauci AS. Multifactorial nature of human immunodeficiency virus disease: Implications for therapy. Science 262:1011–1018;1993.

    Google Scholar 

  14. Flory E, Hoffmeyer A, Smola U, Rapp UR, Bruder JT. Raf-1 kinase targets GA-binding protein in transcriptional regulation of the human immunodeficiency virus type 1 promoter. J Virol 70:2260–2268;1996.

    Google Scholar 

  15. Folguiera L, Algeciras A, McMorran WS, Bren GD, Paya CV. The Ras-Raf pathway is activated in human immunodeficiency virus infected monocytes and participates in the activation of NFκB. J Virol 70:2332–2338;1996.

    Google Scholar 

  16. Goodenow M, Huet T, Saurin W, Kwok S, Shinsky J, Wain-Hobson S. HIV-1 isolates are rapidly evolving quasispecies: Evidence for viral mixtures and preferred nucleotide substitutions. J AIDS 2:344–352;1989.

    Google Scholar 

  17. Hellerstein MK, McCunes JM. T cell turnover in HIV-1 disease. Immunity 7:583–589;1997.

    Google Scholar 

  18. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373:123–126;1995.

    Google Scholar 

  19. Hughes SH, Shank PR, Spector DH, Kung HJ, Bishop JM, Varmus HE, Vogt PK, Breitman ML. Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites. Cell 15:1397–1410;1978.

    Google Scholar 

  20. Jones KA, Peterlin BM. Control of RNA initiation and elongation at the HIV-1 promoter. Annu Rev Biochem 63:717–743;1994.

    Google Scholar 

  21. Ju G, Skalka AM. Nucleotide sequence analysis of the long terminal repeat (LTR) of avian retroviruses: Structural similarities with transposable elements. Cell 22:379–386;1980.

    Google Scholar 

  22. Kadonaga JT. Purification of sequence-specific binding proteins by DNA affinity chromatography. Sauer RT, ed. Methods in Enzymology Protein-DNA Interactions. San Diego, Academic Press, vol 208, 10–23;1991.

    Google Scholar 

  23. Kadosh D, Struhl K. Repression by Ume6 involves recruitment of a complex containing Sin3 corpressor and Rpd3 histone deacetylase to target promoters. Cell 89:365–371;1997.

    Google Scholar 

  24. Koken SE, van Wamel JL, Goudsmit J, Berkhout B, Geelen JL. Natural variants of the HIV-1 long terminal repeat: Analysis of promoters with duplicated DNA regulatory motifs. Virology 191:968–972;1992.

    Google Scholar 

  25. Koken SE, VanWamel JL, Goudsmit J, Berkhout B, Geelen JL. Functional analysis of the ACTGCTGA sequence motif in the human immunodeficiency virus type-1 long terminal repeat promoter. J Biomed Sci 1:83–92;1994.

    Google Scholar 

  26. LaMarco K, Thompson CC, Byers BP, Walton EM, McKnight SL. Identification of Ets- and notch-related subunits in GA binding protein. Science 25:789–792;1991.

    Google Scholar 

  27. Meyerhans A, Cheynier R, Albert J, Seth M, Kwok S, Sninsky J, Morfeldt-Manson L, Asjo B, Wain-Hobson S. Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Cell 58:901–910;1989.

    Google Scholar 

  28. Moodie SA, Wolfman A. The 3 Rs of life: Ras, Raf and growth regulation. Trends Genet 10:44–48;1994.

    Google Scholar 

  29. Moretti P, Freeman K, Coodly L, Shore D. Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev 8:2257–2269;1994.

    Google Scholar 

  30. Myers G. Human Retroviruses and AIDS. Los Alamos, Los Alamos National Library, 1996.

    Google Scholar 

  31. Pantaleo G, Fauci AS. Immunopathogenesis of HIV infection. Annu Rev Microbiol 50:825–853;1996.

    Google Scholar 

  32. Rundlett SE, Carmen AA, Suka N, Turner BM, Grunstein M. Transcriptional repression by UME6 involves deacetylation of lysine 5 of histone H4 by RPD3. Nature 392:831–835;1998.

    Google Scholar 

  33. Shank RP, Hughes SH, Kung HJ, Majors JE, Quintrell N, Guntaka RV, Bishoop JM, Varmus HE. Mapping unintegrated avian sarcoma virus DNA: Termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA. Cell 15:1383–1395;1978.

    Google Scholar 

  34. Shore D. RAP1: A protean regulator in yeast. Trends Genet 10:408–412;1994.

    Google Scholar 

  35. Taylor JM. DNA intermediates of avian RNA tumor viruses. Curr Top Microbiol Immuno 87:23–41;1979.

    Google Scholar 

  36. Thompson CC, Brown TA, McKnight SL. Convergence of Ets-and Notch-related motifs in a heteromeric DNA binding complex. Science 253:762–768;1991.

    Google Scholar 

  37. Treitel MA, Carlson M. Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein. Proc Natl Acad Sci USA 92:3132–3136;1995.

    Google Scholar 

  38. Wain-Hobson S. Down or out in blood and lymph? Nature 387:123–124;1997.

    Google Scholar 

  39. Wain-Hobson S. Running the gamut of retroviral variation. Trends Microbiol 4:135–141;1996.

    Google Scholar 

  40. Wei X, Ghosh SK, Taylor ME, Johnson VA, Emini EA, Deutsch P, Lifson JD, Bonhoeffer S, Nowak MA, Hahn BH, Saag MS, Shaw GM. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 373:117–122;1995.

    Google Scholar 

  41. Yammamato T, deCrombrugghe B, Pastan I. Identification of a functional promoter in the long terminal repeat of Rous sarcoma virus. Cell 22:787–797;1980.

    Google Scholar 

  42. Zhang L, Huang Y, Yuan H, Chen BK, Ip J, HO DD. Genotypic and phenotypic characterization of long terminal repeat sequences from long-term survivors of human immunodeficiency virus type 1 infection. J Virol 71:5608–5613;1997.

    Google Scholar 

  43. Zhang L, Huang Y, Yuan H, Chen BK, Ip J, Ho DD. Identification of a replication-competent pathogenic human immunodeficiency virus type 1 with a duplication in the TCF-1 alpha region but lacking NF-kappaB binding sites. J Virol 71:1651–1656;1997.

    Google Scholar 

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Authors and Affiliations

  1. B.C. Center for Excellence in HIV/AIDS, St. Paul's Hospital, Vancouver, B.C., Canada

    Mario Clemente Estable & Michael V. O'Shaughnessy

  2. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, 2146, Health Sciences Mall, V6T 1Z3, Vancouver, B.C., Canada

    Mario Clemente Estable, Martin Hirst, Brendan Bell & Ivan Sadowski

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  1. Mario Clemente Estable
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  4. Michael V. O'Shaughnessy
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Estable, M.C., Hirst, M., Bell, B. et al. Purification of RBF-2, a transcription factor with specificity for the most conservedcis-element of naturally occurring HIV-1 LTRs. J Biomed Sci 6, 320–332 (1999). https://doi.org/10.1007/BF02253521

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  • DOI: https://doi.org/10.1007/BF02253521

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