Journal of Biomedical Science

, Volume 8, Issue 5, pp 430–436 | Cite as

Binding of phage-displayed HIV-1 tat to TAR RNA in the presence of cyclin T1

  • Gesa Jonas
  • Silke Hoffmann
  • Dieter Willbold
Original Paper
Received:
Accepted:

Abstract

The transactivator protein (Tat) of the human immunodeficiency virus (HIV) is a key regulatory protein in the viral replication cycle. Together with cellular cyclin T1 and an RNA element (transactivation response; TAR) located at the 5′ end of all viral transcripts, it forms a ternary complex that ultimately enhances the expression of all viral genes. In this ternary complex, cyclin T1 interacts directly with Tat and TAR. The presence of cyclin T1 is essential for high TAR RNA affinity and specificity of Tat. To study protein-protein and protein-RNA interaction, we developed a phage display system that displays functional Tat on the surface of bacteriophage M13. The addition of recombinant cyclin T1 to the selections yielded a phage display system that mirrors all binding properties of the cyclin T1-Tat-TAR complex known from cell assays and biochemical studies. Phage-displayed Tat protein as well as the cyclin T1 are fully functional. The relative binding capabilities of wild-type- and mutant Tat-displaying phages show that the presence of cyclin T1 significantly reduces the importance of basic residues in the basic sequence region of Tat for its binding to TAR.

Key Words

HIV-1 Transactivator Cyclin T1 Phage display Protein-RNA interactions 
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Bieniasz PD, Grdina TA, Bogerd HP, Cullen BR. Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat. EMBO J 17:7056–7065;1998.Google Scholar
  2. 2.
    Burd CG, Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science 265:615–621;1994.Google Scholar
  3. 3.
    Churcher M, Lamont JC, Hamy F, Dingwall C, Green SM, Lowe AD, Butler JG, Gait MJ, 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 230:90–110;1993.Google Scholar
  4. 4.
    Cullen BR. Trans-activation of human immunodeficiency occurs via a bimodal mechanism. Cell 46:973–982;1986.Google Scholar
  5. 5.
    Dayton AI, Sodrowski JG, Rosen CA, Goh WC, Haseltine WA. The trans-activator gene of the human T cell lymphotropic virus type III is required for replication. Cell 44:941–947;1986.Google Scholar
  6. 6.
    Emerman M, Malim MH. HIV-1 regulatory/accessory genes: Keys to unraveling viral and host cell biology. Science 280:1880–1884;1998.Google Scholar
  7. 7.
    Ensoli B, Buonaguro L, Barillari G, Fiorelli V, Gendelman R, Morgan RA, Wingfield P, Gallo RC. Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation. J Virol 67:277–287;1993.Google Scholar
  8. 8.
    Fisher AG, Feinberg MB, Josephs SB, Harper ME, Marselle LM, Reyes G, Gonda MA, Aldovini A, Debouck C, Gallo RC, Wong-Staal F. The trans-activator gene of the HTLV-III is essential for virus replication. Nature 320:367–371;1986.Google Scholar
  9. 9.
    Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55:1189–1193;1988.Google Scholar
  10. 10.
    Fujinaga K, Cujec TP, Peng J, Garriga J, Price DH, Grana X, Peterlin MB. The ability of positive transcription elongation factor B to transactivate human immunodeficiency virus transcription depends on a functional kinase domain, cyclin T1, and Tat. J Virol 72:7154–7159;1998.Google Scholar
  11. 11.
    Hoffmann S, Willbold D. A selection system to study protein-RNA interactions: Functional display of HIV-1 Tat protein on filamentous bacteriophage M13. Biochem Biophys Res Commun 235:806–811;1997.Google Scholar
  12. 12.
    Jones KA. Taking a new TAK on tat transactivation. Genes Dev 11:2593–2599;1997.Google Scholar
  13. 13.
    Jones KA, Peterlin BM. Control of RNA initiation and elongation at the HIV-1 promoter. Annu Rev Biochem 63:717–743;1994.Google Scholar
  14. 14.
    Krebber A, Burmester J, Plückthun A. Inclusion of upstream transcriptional terminator in phage display vectors abolishes background expression of toxic fusions with coat protein g3p. Gene 178:71–74;1996.Google Scholar
  15. 15.
    Lazinski D, Grzadzielska E, Das A. Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell 59:207–218;1989.Google Scholar
  16. 16.
    Luo Y, Madore S, Parslow T, Cullen B, Peterlin BM. Functional analysis of interaction between Tat and the trans-activation response element of human immunodeficiency virus type 1 in cells. J Virol 67:5617–5622;1993.Google Scholar
  17. 17.
    Puglisi JD, Chen L, Blanchard S, Frankel AD. Solution structure of a bovine immunodeficiency virus Tat-TAR peptide-RNA complex. Science 270:1200–1203;1995.Google Scholar
  18. 18.
    Seewald MJ, Metzger AU, Willbold D, Rösch P, Sticht H. Structural model of the HIV-1 Tat(46–58)-TAR complex. J Biomol Struct Dyn 16:683–692;1998.Google Scholar
  19. 19.
    Sticht H, Willbold D, Ejchart A, Rosin-Arbesfeld R, Yaniv A, Gazit A, Rösch P. Trifluoroethanol stabilizes a helix-turn-helix motif in equine infectious-anemia-virus trans-activator protein. Eur J Biochem 225:855–861;1994.Google Scholar
  20. 20.
    Tan R, Frankel AD. Costabilization of peptide and RNA structure in an HIV Rev peptide-RRE complex. Biochemistry 33:14579–14585;1994.Google Scholar
  21. 21.
    Tan R, Frankel AD. Structural variety of arginine-rich RNA-binding peptides. Proc Natl Acad Sci USA 92:5282–5286;1995.Google Scholar
  22. 22.
    Wei P, Garber ME, Fang S-M, Fisher WH, Jones KA. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92:451–462;1998.Google Scholar
  23. 23.
    Willbold D, Rosin-Arbesfeld R, Sticht H, Frank R, Rösch P. Structure of the equine infectious anemia virus Tat protein. Science 264:1584–1587;1994.Google Scholar
  24. 24.
    Ye X, Kumar RA, Patel DJ. Molecular recognition in the bovine immunodeficiency virus Tat peptide-TAR RNA complex. Chem Biol 2:827–832;1995.Google Scholar

Copyright information

© National Science Council 2001

Authors and Affiliations

  • Gesa Jonas
    • 2
  • Silke Hoffmann
    • 1
  • Dieter Willbold
    • 2
  1. 1.Lehrstuhl für BiopolymereUniversität BayreuthBayreuthDeutschland
  2. 2.Institut für Molekulare BiotechnologieStrukturelle und Evolutive BiochemieJenaGermany

Personalised recommendations