Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Aug 25;22(16):3365–3372. doi: 10.1093/nar/22.16.3365

Identification of a novel HIV-1 TAR RNA bulge binding protein.

B Baker 1, M Muckenthaler 1, E Vives 1, A Blanchard 1, M Braddock 1, W Nacken 1, A J Kingsman 1, S M Kingsman 1
PMCID: PMC523730  PMID: 8078772

Abstract

The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome. Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem. TAR is located at the 5' end of all viral RNAs. In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop. However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors. A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors. We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR. The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae. BBP is an effective competitive inhibitor of Tat binding to TAR in vitro. Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo.

Full text

PDF
3365

Images in this article

Selected References

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

  1. Alonso A., Derse D., Peterlin B. M. Human chromosome 12 is required for optimal interactions between Tat and TAR of human immunodeficiency virus type 1 in rodent cells. J Virol. 1992 Jul;66(7):4617–4621. doi: 10.1128/jvi.66.7.4617-4621.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blanchard A. D., Powell R., Braddock M., Kingsman A. J., Kingsman S. M. An adenosine at position 27 in the human immunodeficiency virus type 1 trans-activation response element is not critical for transcriptional or translational activation by Tat. J Virol. 1992 Nov;66(11):6769–6772. doi: 10.1128/jvi.66.11.6769-6772.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Braddock M., Chambers A., Wilson W., Esnouf M. P., Adams S. E., Kingsman A. J., Kingsman S. M. HIV-1 TAT "activates" presynthesized RNA in the nucleus. Cell. 1989 Jul 28;58(2):269–279. doi: 10.1016/0092-8674(89)90841-6. [DOI] [PubMed] [Google Scholar]
  4. Braddock M., Powell R., Blanchard A. D., Kingsman A. J., Kingsman S. M. HIV-1 TAR RNA-binding proteins control TAT activation of translation in Xenopus oocytes. FASEB J. 1993 Jan;7(1):214–222. doi: 10.1096/fasebj.7.1.8422967. [DOI] [PubMed] [Google Scholar]
  5. Carrier F., Gatignol A., Hollander M. C., Jeang K. T., Fornace A. J., Jr Induction of RNA-binding proteins in mammalian cells by DNA-damaging agents. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1554–1558. doi: 10.1073/pnas.91.4.1554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Churcher M. J., Lamont C., Hamy F., Dingwall C., Green S. M., Lowe A. D., Butler J. G., Gait M. J., Karn J. High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region. J Mol Biol. 1993 Mar 5;230(1):90–110. doi: 10.1006/jmbi.1993.1128. [DOI] [PubMed] [Google Scholar]
  7. Colvin R. A., Garcia-Blanco M. A. Unusual structure of the human immunodeficiency virus type 1 trans-activation response element. J Virol. 1992 Feb;66(2):930–935. doi: 10.1128/jvi.66.2.930-935.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cullen B. R. Does HIV-1 Tat induce a change in viral initiation rights? Cell. 1993 May 7;73(3):417–420. doi: 10.1016/0092-8674(93)90126-b. [DOI] [PubMed] [Google Scholar]
  9. Cullen B. R., Greene W. C. Regulatory pathways governing HIV-1 replication. Cell. 1989 Aug 11;58(3):423–426. doi: 10.1016/0092-8674(89)90420-0. [DOI] [PubMed] [Google Scholar]
  10. Cullen B. R. Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol Rev. 1992 Sep;56(3):375–394. doi: 10.1128/mr.56.3.375-394.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Delling U., Reid L. S., Barnett R. W., Ma M. Y., Climie S., Sumner-Smith M., Sonenberg N. Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure. J Virol. 1992 May;66(5):3018–3025. doi: 10.1128/jvi.66.5.3018-3025.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Desai K., Loewenstein P. M., Green M. Isolation of a cellular protein that binds to the human immunodeficiency virus Tat protein and can potentiate transactivation of the viral promoter. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8875–8879. doi: 10.1073/pnas.88.20.8875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dingwall C., Ernberg I., Gait M. J., Green S. M., Heaphy S., Karn J., Lowe A. D., Singh M., Skinner M. A. HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure. EMBO J. 1990 Dec;9(12):4145–4153. doi: 10.1002/j.1460-2075.1990.tb07637.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Frankel A. D. Peptide models of the Tat-TAR protein-RNA interaction. Protein Sci. 1992 Dec;1(12):1539–1542. doi: 10.1002/pro.5560011202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gait M. J., Karn J. RNA recognition by the human immunodeficiency virus Tat and Rev proteins. Trends Biochem Sci. 1993 Jul;18(7):255–259. doi: 10.1016/0968-0004(93)90176-n. [DOI] [PubMed] [Google Scholar]
  17. Gatignol A., Buckler-White A., Berkhout B., Jeang K. T. Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science. 1991 Mar 29;251(5001):1597–1600. doi: 10.1126/science.2011739. [DOI] [PubMed] [Google Scholar]
  18. Gatignol A., Buckler C., Jeang K. T. Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen. Mol Cell Biol. 1993 Apr;13(4):2193–2202. doi: 10.1128/mcb.13.4.2193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gunnery S., Green S. R., Mathews M. B. Tat-responsive region RNA of human immunodeficiency virus type 1 stimulates protein synthesis in vivo and in vitro: relationship between structure and function. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11557–11561. doi: 10.1073/pnas.89.23.11557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jaeger J. A., Tinoco I., Jr An NMR study of the HIV-1 TAR element hairpin. Biochemistry. 1993 Nov 23;32(46):12522–12530. doi: 10.1021/bi00097a032. [DOI] [PubMed] [Google Scholar]
  21. Jeang K. T., Chun R., Lin N. H., Gatignol A., Glabe C. G., Fan H. In vitro and in vivo binding of human immunodeficiency virus type 1 Tat protein and Sp1 transcription factor. J Virol. 1993 Oct;67(10):6224–6233. doi: 10.1128/jvi.67.10.6224-6233.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kashanchi F., Piras G., Radonovich M. F., Duvall J. F., Fattaey A., Chiang C. M., Roeder R. G., Brady J. N. Direct interaction of human TFIID with the HIV-1 transactivator tat. Nature. 1994 Jan 20;367(6460):295–299. doi: 10.1038/367295a0. [DOI] [PubMed] [Google Scholar]
  23. Madore S. J., Cullen B. R. Genetic analysis of the cofactor requirement for human immunodeficiency virus type 1 Tat function. J Virol. 1993 Jul;67(7):3703–3711. doi: 10.1128/jvi.67.7.3703-3711.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Marciniak R. A., Garcia-Blanco M. A., Sharp P. A. Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1990 May;87(9):3624–3628. doi: 10.1073/pnas.87.9.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Masuda T., Harada S. Modulation of host cell nuclear proteins that bind to HIV-1 trans-activation-responsive element RNA by phorbol ester. Virology. 1993 Feb;192(2):696–700. doi: 10.1006/viro.1993.1091. [DOI] [PubMed] [Google Scholar]
  26. Mellor J., Dobson M. J., Roberts N. A., Tuite M. F., Emtage J. S., White S., Lowe P. A., Patel T., Kingsman A. J., Kingsman S. M. Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene. 1983 Sep;24(1):1–14. doi: 10.1016/0378-1119(83)90126-9. [DOI] [PubMed] [Google Scholar]
  27. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Michnicka M. J., Harper J. W., King G. C. Selective isotopic enrichment of synthetic RNA: application to the HIV-1 TAR element. Biochemistry. 1993 Jan 19;32(2):395–400. doi: 10.1021/bi00053a002. [DOI] [PubMed] [Google Scholar]
  29. Nagai K., Thøgersen H. C. Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli. 1984 Jun 28-Jul 4Nature. 309(5971):810–812. doi: 10.1038/309810a0. [DOI] [PubMed] [Google Scholar]
  30. Ohana B., Moore P. A., Ruben S. M., Southgate C. D., Green M. R., Rosen C. A. The type 1 human immunodeficiency virus Tat binding protein is a transcriptional activator belonging to an additional family of evolutionarily conserved genes. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):138–142. doi: 10.1073/pnas.90.1.138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Riordan F. A., Bhattacharyya A., McAteer S., Lilley D. M. Kinking of RNA helices by bulged bases, and the structure of the human immunodeficiency virus transactivator response element. J Mol Biol. 1992 Jul 20;226(2):305–310. doi: 10.1016/0022-2836(92)90947-i. [DOI] [PubMed] [Google Scholar]
  32. Rounseville M. P., Kumar A. Binding of a host cell nuclear protein to the stem region of human immunodeficiency virus type 1 trans-activation-responsive RNA. J Virol. 1992 Mar;66(3):1688–1694. doi: 10.1128/jvi.66.3.1688-1694.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sheline C. T., Milocco L. H., Jones K. A. Two distinct nuclear transcription factors recognize loop and bulge residues of the HIV-1 TAR RNA hairpin. Genes Dev. 1991 Dec;5(12B):2508–2520. doi: 10.1101/gad.5.12b.2508. [DOI] [PubMed] [Google Scholar]
  35. Swanson M. S., Dreyfuss G. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities. Mol Cell Biol. 1988 May;8(5):2237–2241. doi: 10.1128/mcb.8.5.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Weeks K. M., Crothers D. M. RNA recognition by Tat-derived peptides: interaction in the major groove? Cell. 1991 Aug 9;66(3):577–588. doi: 10.1016/0092-8674(81)90020-9. [DOI] [PubMed] [Google Scholar]
  37. Wu F., Garcia J., Sigman D., Gaynor R. tat regulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185. Genes Dev. 1991 Nov;5(11):2128–2140. doi: 10.1101/gad.5.11.2128. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES