Skip to main content

Advertisement

SpringerLink
Log in

Interaction of hepatitis B virus X protein with damaged DNA-binding protein p127: Structural analysis and identification of antagonists

  • Original Paper
  • Published:
Journal of Biomedical Science

Abstract

The hepatitis B virus X protein is a multifunctional protein that is essential for natural infection and has also been implicated in liver cancer development. Previous studies have identified the DDB1 subunit of the damaged-DNA binding complex as a critical partner of X protein in the infection process, X-mediated cytotoxicity and stability of the viral protein. Here, we investigated the structural and functional constraints of X-DDB1 interaction using various mutational analyses. Our data show that the interaction interface of X with DDB1 is confined to a 15-residue epitope. All substitutions responsible for loss of binding mapped to this core-binding domain. In contrast, a marked increase in affinity for DDB1 resulted from substitutions at clustered positions lying close to the DDB1-binding epitope and correlated with loss of apoptotic potential. Selection of mutations in DDB1 that partially rescue the binding defect of an X mutant gave further insight into the contacts established between the two proteins. Importantly, both the core-binding domain of X and the gain-of-affinity X mutants inhibited DDB1-mediated stabilization of wild-type X protein. These X protein derivatives thus provide the basis for the development of therapeutic agents that antagonize X function through competitive inhibition of X-DDB1 interaction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Adams PD, Sellers WR, Sharma SK, Wu AD, Nalin CM, Kaelin WG Jr. Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors. Mol Cell Biol 16:6623–6633;1996.

    PubMed  Google Scholar 

  2. Barnabas S, Hai T, Andrisani OM. The hepatitis B virus X protein enhances the DNA binding potential and transcription efficacy of bZip transcription factors. J Biol Chem 272:20684–20690;1997.

    Google Scholar 

  3. Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan. Lancet ii:1129–1133;1981.

    Article  Google Scholar 

  4. Benn J, Schneider RJ. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proc Natl Acad Sci USA 91:10350–10354;1994.

    PubMed  Google Scholar 

  5. Benn J, Su F, Doria M, Schneider RJ. Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases. J Virol 70:4978–4985;1996.

    PubMed  Google Scholar 

  6. Bergametti F, Prigent S, Luber B, Benoit A, Tiollais P, Sarasin A, Transy C. The proapoptotic effect of hepatitis B virus HBx protein correlates with its transactivation activity in stably transfected cell lines. Oncogene 18:2860–2871;1999.

    PubMed  Google Scholar 

  7. Bergametti F, Sitterlin D, Transy C. Turnover of hepatitis B virus X protein is regulated by damaged DNA-binding complex. J Virol 76:6495–6501;2002.

    PubMed  Google Scholar 

  8. Bouchard MJ, Wang LH, Schneider RJ. Calcium signaling by HBx protein in hepatitis B virus DNA replication. Science 294:2376–2378;2001.

    Article  PubMed  Google Scholar 

  9. Buendia MA. Hepatitis B virus and hepatocellular carcinoma. Adv Cancer Res 59:167–226;1992.

    PubMed  Google Scholar 

  10. Chang SF, Netter HJ, Hildt E, Schuster R, Schaefer S, Hsu YC, Rang A, Will H. Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame. J Virol 75:161–170;2001.

    Article  PubMed  Google Scholar 

  11. Chen HS, Kaneko S, Girones R, Anderson RW, Hornbuckle WE, Tennant BC, Cote PJ, Gerin JL, Purcell RH, Miller RH. The wood-chuck hepatitis virus X gene is important for establishment of virus infection in wood-chucks. J Virol 67:1218–1226;1993.

    PubMed  Google Scholar 

  12. Chen YN, Sharma SK, Ramsey TM, Jiang L, Martin MS, Baker K, Adams PD, Bair KW, Kaelin WG Jr. Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists. Proc Natl Acad Sci USA 96:4325–4329;1999.

    Google Scholar 

  13. Doria M, Klein N, Lucito R, Schneider RJ. The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors. EMBO J 14:4747–4757;1995.

    PubMed  Google Scholar 

  14. Fischer M, Runkel L, Schaller H. HBx protein of hepatitis B virus interacts with the C-terminal portion of a novel human proteasome alpha-subunit. Virus Genes 10:99–102;1995.

    Article  PubMed  Google Scholar 

  15. Forgues M, Marrogi AJ, Spillare EA, Wu CG, Yang Q, Yoshida M, Wang XW. Interaction of the hepatitis B virus X protein with the Crm1-dependent nuclear export pathway. J Biol Chem 276:22797–22803;2001.

    PubMed  Google Scholar 

  16. Galibert F, Mandart E, Fitoussi F, Tiollais P, Charnay P. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned inE. coli. Nature 281:646–650;1979.

    Article  PubMed  Google Scholar 

  17. Girones R, Cote PJ, Hornbuckle WE, Tennant BC, Gerin JL, Purcell RH, Miller RH. Complete nucleotide sequence of a molecular clone of woodchuck hepatitis virus that is infectious in the natural host. Proc Natl Acad Sci USA 86:1846–1849;1989.

    PubMed  Google Scholar 

  18. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816;1993.

    Article  PubMed  Google Scholar 

  19. Haviv I, Shamay M, Doitsh G, Shaul Y. Hepatitis B virus pX targets TFIIB in transcription coactivation. Mol Cell Biol 18:1562–1569;1998.

    PubMed  Google Scholar 

  20. Haviv I, Vaizel D, Shaul Y. pX, the HBV-encoded coactivator, interacts with components of the transcription machinery and stimulates transcription in a TAF-independent manner. EMBO J 15:3413–3420;1996.

    PubMed  Google Scholar 

  21. Hollenberg SM, Sternglanz R, Cheng PF, Weintraub H. Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system. Mol Cell Biol 15:3813–3822;1995.

    PubMed  Google Scholar 

  22. Hu Z, Zhang Z, Doo E, Coux O, Goldberg AL, Liang TJ. Hepatitis B virus X protein is both a substrate and a potential inhibitor of the proteasome complex. J Virol 73:7231–7240;1999.

    PubMed  Google Scholar 

  23. Klein NP, Bouchard MJ, Wang LH, Kobarg C, Schneider RJ. Src kinases involved in hepatitis B virus replication. EMBO J 18:5019–5027;1999.

    PubMed  Google Scholar 

  24. Lanford RE, Chavez D, Brasky KM, Burns RB 3rd, Rico-Hesse R. Isolation of a hepadnavirus from the woolly monkey, a New World primate. Proc Natl Acad Sci USA 95:5757–5761;1998.

    PubMed  Google Scholar 

  25. Lee JO, Russo AA, Pavletich NP. Structure of the retinoblastoma tumour-suppressor pocket domain bound to a peptide from HPV E7. Nature 391:859–865;1998.

    PubMed  Google Scholar 

  26. Lee TH, Elledge SJ, Butel JS. Hepatitis B virus X protein interacts with a probable cellular DNA repair protein. J Virol 69:1107–1114;1995.

    PubMed  Google Scholar 

  27. Lin-Marq N, Bontron S, Leupin O, Strubin M. Hepatitis B virus X protein interferes with cell viability through interaction with the p127-kDa UV-damaged DNA-binding protein. Virology 287:266–274;2001.

    Article  PubMed  Google Scholar 

  28. Murakami S. Hepatitis B virus X protein: A multifunctional viral regulator. J Gastroenterol 36:651–660;2001.

    Article  PubMed  Google Scholar 

  29. Neuwald AF, Poleksic A. PSI-BLAST searches using hidden Markov models of structural repeats: Prediction of an unusual sliding DNA clamp and of beta-propellers in UV-damaged DNA-binding protein. Nucleic Acids Res 28:3570–3580;2000.

    PubMed  Google Scholar 

  30. Qadri I, Conaway JW, Conaway RC, Schaack J, Siddiqui A. Hepatitis B virus transactivator protein, HBx, associates with the components of TFIIH and stimulates the DNA helicase activity of TFIIH. Proc Natl Acad Sci USA 93:10578–10583;1996.

    Article  PubMed  Google Scholar 

  31. Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B. A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032;1999.

    Article  PubMed  Google Scholar 

  32. Rossner MT. Hepatitis B virus X-gene product: A promiscuous transcriptional activator. J Med Virol 36:101–117;1992.

    Article  PubMed  Google Scholar 

  33. Schek N, Bartenschlager R, Kuhn C, Schaller H. Phosphorylation and rapid turnover of hepatitis B virus X-protein expressed in HepG2 cells from a recombinant vaccinia virus. Oncogene 6:1735–1744;1991.

    PubMed  Google Scholar 

  34. Schödel F, Sprengel R, Weimer T, Fernholz D, Schneider R, Will H. Animal hepatitis B viruses. In: Klein G, ed. Advances in Viral Oncology, Vol. 8. New York, Raven Press, 73–102; 1989.

    Google Scholar 

  35. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: Delivery of a biologically active protein into the mouse. Science 285:1569–1572;1999.

    Article  PubMed  Google Scholar 

  36. Schwarze SR, Hruska KA, Dowdy SF. Protein transduction: Unrestricted delivery into all cells? Trends Cell Biol 10:290–295;2000.

    Article  PubMed  Google Scholar 

  37. Sitterlin D, Bergametti F, Tiollais P, Tennant BC, Transy C. Correct binding of viral X protein to UVDDB-p127 cellular protein is critical for efficient infection by hepatitis B viruses. Oncogene 19:4427–4431;2000.

    Article  PubMed  Google Scholar 

  38. Sitterlin D, Bergametti F, Transy C. UVDDB p127-binding modulates activities and intracellular distribution of hepatitis B virus X protein. Oncogene 19:4417–4426;2000.

    Article  PubMed  Google Scholar 

  39. Sitterlin D, Lee TH, Prigent S, Tiollais P, Butel JS, Transy C. Interaction of the UV-damaged DNA-binding protein with hepatitis B virus X protein is conserved among mammalian hepadnaviruses and restricted to transactivation-proficient X-insertion mutants. J Virol 71:6194–6199;1997.

    PubMed  Google Scholar 

  40. Testut P, Renard CA, Terradillos O, Vitvitski-Trepo L, Tekaia F, Degott C, Blake J, Boyer B, Buendia MA. A new hepadnavirus endemic in arctic ground squirrels in Alaska. J Virol 70:4210–4219;1996.

    PubMed  Google Scholar 

  41. Truant R, Antunovic J, Greenblatt J, Prives C, Cromlish JA. Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation. J Virol 69:1851–1859;1995.

    PubMed  Google Scholar 

  42. Ueda H, Ullrich SJ, Gangemi JD, Kappel CA, Ngo L, Feitelson MA, Jay G. Functional inactivation but not structural mutation of p53 causes liver cancer. Nat Genet 9:41–47;1995.

    Article  PubMed  Google Scholar 

  43. Zoulim F, Saputelli J, Seeger C. Woodchuck hepatitis virus X protein is required for viral infection in vivo. J Virol 68:2026–2030;1994.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. UREG/INSERM U163, Institut Pasteur, 28 rue du Dr. Roux, F-75724, Paris Cedex 15, France

    Françoise Bergametti, Julie Bianchi &  Catherine Transy

Authors
  1. Françoise Bergametti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julie Bianchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Catherine Transy
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bergametti, F., Bianchi, J. & Transy, C. Interaction of hepatitis B virus X protein with damaged DNA-binding protein p127: Structural analysis and identification of antagonists. J Biomed Sci 9, 706–715 (2002). https://doi.org/10.1007/BF02254999

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02254999

Key Words

Search

Navigation