Skip to main content

Advertisement

SpringerLink
Log in

Conditional expression of a dominant-negative form of Epstein-Barr virus (EBV) nuclear antigen EBNALP inhibits EBV-positive lymphoblastoid cell growth

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that transforms primary B lymphocytes, yielding lymphoblastoid cell lines (LCLs). EBV-encoded nuclear antigen 2 (EBNA2) and EBV-encoded nuclear antigen leader protein (EBNALP) are the first viral products expressed after EBV infection of primary B lymphocytes and are essential for EBV-induced B-lymphocyte growth transformation. EBNA2 functions as a transcriptional activator of viral and cellular genes, with EBNALP as a coactivator for EBNA2-mediated transcriptional activation. We previously reported that mutant EBNALP with a C-terminal 10-amino-acid truncation loses the ability to coactivate, and has a dominant-negative effect on wild-type-EBNALP-mediated coactivation. However, the functional relevance of EBNALP in maintenance of LCL cell growth has not been investigated. To address this, we have constructed LCL-derived cell clones in which this dominant-negative form of EBNALP (DNLP) is conditionally expressed by the Cre-loxP system. We used these cells to evaluate the effect of DNLP expression on EBV-induced cell proliferation. After drug treatment, the DNLP-expressing LCL clones showed reduced cell proliferation and viability. These results indicate that EBNALP is critical for maintaining LCL growth and EBV-induced cell proliferation.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Longnecker R, Kieff E, Cohen J (2013) Epstein-Barr virus. In: Knipe D, Howley P (eds) Fields virology, vol 2, 6th edn. Lippincott Williams and Wilkins, Philadelphia, pp 1898–1959

    Google Scholar 

  2. Alfieri C, Birkenbach M, Kieff E (1991) Early events in Epstein-Barr virus infection of human B lymphocytes. Virology 181(2):595–608

    Article  CAS  Google Scholar 

  3. Wang F, Tsang SF, Kurilla MG, Cohen JI, Kieff E (1990) Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1. J Virol 64(7):3407–3416

    Article  CAS  Google Scholar 

  4. Wang F, Gregory C, Sample C, Rowe M, Liebowitz D, Murray R, Rickinson A, Kieff E (1990) Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J Virol 64(5):2309–2318

    Article  CAS  Google Scholar 

  5. Harada S, Kieff E (1997) Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. J Virol 71(9):6611–6618

    Article  CAS  Google Scholar 

  6. Sinclair AJ, Palmero I, Peters G, Farrell PJ (1994) EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by Epstein-Barr virus. EMBO J 13(14):3321–3328

    Article  CAS  Google Scholar 

  7. Mannick JB, Cohen JI, Birkenbach M, Marchini A, Kieff E (1991) The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation. J Virol 65(12):6826–6837

    Article  CAS  Google Scholar 

  8. Szymula A, Palermo RD, Bayoumy A, Groves IJ, Ba Abdullah M, Holder B, White RE (2018) Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naive B cells, and facilitates recruitment of transcription factors to the viral genome. PLoS Pathog 14(2):e1006890. https://doi.org/10.1371/journal.ppat.1006890

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Peng CW, Xue Y, Zhao B, Johannsen E, Kieff E, Harada S (2004) Direct interactions between Epstein-Barr virus leader protein LP and the EBNA2 acidic domain underlie coordinate transcriptional regulation. Proc Natl Acad Sci USA 101(4):1033–1038. https://doi.org/10.1073/pnas.0307808100

    Article  CAS  PubMed  Google Scholar 

  10. Kanegae Y, Lee G, Sato Y, Tanaka M, Nakai M, Sakaki T, Sugano S, Saito I (1995) Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res 23(19):3816–3821

    Article  CAS  Google Scholar 

  11. Tsukiyama-Kohara K, Tone S, Maruyama I, Inoue K, Katsume A, Nuriya H, Ohmori H, Ohkawa J, Taira K, Hoshikawa Y, Shibasaki F, Reth M, Minatogawa Y, Kohara M (2004) Activation of the CKI-CDK-Rb-E2F pathway in full genome hepatitis C virus-expressing cells. J Biol Chem 279(15):14531–14541. https://doi.org/10.1074/jbc.M312822200

    Article  CAS  PubMed  Google Scholar 

  12. Kanegae Y, Takamori K, Sato Y, Lee G, Nakai M, Saito I (1996) Efficient gene activation system on mammalian cell chromosomes using recombinant adenovirus producing Cre recombinase. Gene 181(1–2):207–212

    Article  CAS  Google Scholar 

  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  CAS  Google Scholar 

  14. Harada S, Yalamanchili R, Kieff E (1998) Residues 231 to 280 of the Epstein-Barr virus nuclear protein 2 are not essential for primary B-lymphocyte growth transformation. J Virol 72(12):9948–9954

    Article  CAS  Google Scholar 

  15. Daum G, Eisenmann-Tappe I, Fries HW, Troppmair J, Rapp UR (1994) The ins and outs of Raf kinases. Trends Biochem Sci 19(11):474–480

    Article  CAS  Google Scholar 

  16. Casamayor A, Morrice NA, Alessi DR (1999) Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo. Biochem J 342(Pt 2):287–292

    Article  CAS  Google Scholar 

  17. Zimmermann S, Moelling K (1999) Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286(5445):1741–1744

    Article  CAS  Google Scholar 

  18. Grossman SR, Johannsen E, Tong X, Yalamanchili R, Kieff E (1994) The Epstein-Barr virus nuclear antigen 2 transactivator is directed to response elements by the J kappa recombination signal binding protein. Proc Natl Acad Sci USA 91(16):7568–7572

    Article  CAS  Google Scholar 

  19. Portal D, Zhao B, Calderwood MA, Sommermann T, Johannsen E, Kieff E (2011) EBV nuclear antigen EBNALP dismisses transcription repressors NCoR and RBPJ from enhancers and EBNA2 increases NCoR-deficient RBPJ DNA binding. Proc Natl Acad Sci USA 108(19):7808–7813. https://doi.org/10.1073/pnas.1104991108

    Article  PubMed  Google Scholar 

  20. Ling PD, Peng RS, Nakajima A, Yu JH, Tan J, Moses SM, Yang WH, Zhao B, Kieff E, Bloch KD, Bloch DB (2005) Mediation of Epstein-Barr virus EBNA-LP transcriptional coactivation by Sp100. EMBO J 24(20):3565–3575. https://doi.org/10.1038/sj.emboj.7600820

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kang MS, Kieff E (2015) Epstein-Barr virus latent genes. Exp Mol Med 47:e131. https://doi.org/10.1038/emm.2014.84

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Portal D, Zhou H, Zhao B, Kharchenko PV, Lowry E, Wong L, Quackenbush J, Holloway D, Jiang S, Lu Y, Kieff E (2013) Epstein-Barr virus nuclear antigen leader protein localizes to promoters and enhancers with cell transcription factors and EBNA2. Proc Natl Acad Sci USA 110(46):18537–18542. https://doi.org/10.1073/pnas.1317608110

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Elliott Kieff (Harvard Medical School) for providing IB4 cells and reagents, Dr. Izumu Saito (Institute of Microbial Chemistry) for providing the pCALNL5 and pCANCre plasmids, Dr. Michinori Kohara (Tokyo Metropolitan Institute of Medical Science) for providing the pCAG-PURO-Mer-Cre-Mer plasmid, Dr. Kyoko Yokota (Department of Immunology, National Institute of Infectious Diseases) for providing the antibody against CD11a, and Dr. Misuzu Shimakage for providing the LCLs. We are grateful to Ms. Akie Hirata for excellent technical assistance. This work was partially supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (#14570274, #16590395 and #18K07894).

Author information

Authors and Affiliations

  1. Department of Virology 1, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan

    Shizuko Harada & Masayuki Saijo

Authors
  1. Shizuko Harada
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masayuki Saijo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shizuko Harada.

Ethics declarations

Conflict of interest

We have no conflicts of interest to disclose.

Additional information

Handling Editor: Graciela Andrei.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Harada, S., Saijo, M. Conditional expression of a dominant-negative form of Epstein-Barr virus (EBV) nuclear antigen EBNALP inhibits EBV-positive lymphoblastoid cell growth. Arch Virol 165, 313–320 (2020). https://doi.org/10.1007/s00705-019-04489-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-019-04489-2

Search

Navigation