1887

Abstract

We have previously shown that Epstein–Barr virus (EBV)-encoded EBNA-5 is localized to PML bodies (PODs) in EBV-immortalized lymphoblastoid cell lines (LCLs). Here we have extended our study of the subnuclear localization of EBNA-5 and found a strict co-localization with PML in LCLs and in BL lines with an immunoblastic, LCL-like phenotype. Moreover, GFP–EBNA-5 accumulated in PML bodies upon transfection into LCLs. In contrast, transfection of cell lines of non-immunoblastic origin with an EBNA-5 expression construct showed preferential localization of the protein to the nucleoplasm. Since PML is involved in proteasome-dependent protein degradation, we investigated the total levels and sub-cellular localization of EBNA-5 upon inhibition of proteasome activity. We found that a proteasome inhibitor, MG132, induced the translocation of both endogenous and transfected EBNA-5 to the nucleoli in every cell line tested. The total EBNA-5 protein levels were not affected by the proteasomal block. EBNA-5 forms complexes with heat shock protein Hsp70. The proteasome inhibitor induced a rise in total levels of Hsp70 and dramatically changed its homogeneous nuclear and cytoplasmic distribution into nucleolar and cytoplasmic. This effect was EBNA-5-independent. The nucleolar localization of Hsp70 was enhanced by the presence of EBNA-5, however. EBNA-5 also enhanced the nucleolar translocation of a mutant p53 in a colon cancer line, SW480, treated with MG132. The coordinated changes in EBNA-5 and Hsp70 localization and the effect of EBNA-5 on mutant p53 distribution upon MG132 treatment might reflect the involvement of EBNA-5 in the regulation of intracellular protein trafficking associated with the proteasome-mediated degradation.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-82-2-345
2001-02-01
2020-08-08
Loading full text...

Full text loading...

/deliver/fulltext/jgv/82/2/0820345a.html?itemId=/content/journal/jgv/10.1099/0022-1317-82-2-345&mimeType=html&fmt=ahah

References

  1. Abarzua P., LoSardo J. E., Gubler M. L., Neri A.. 1995; Microinjection of monoclonal antibody PAb421 into human SW480 colorectal carcinoma cells restores the transcription activation function to mutant p53. Cancer Research55:3490–3494
    [Google Scholar]
  2. Alfiery C., Birkenbach M., Kieff E.. 1991; Early events in Epstein–Barr virus infection of human B lymphocytes. Virology181:595–608
    [Google Scholar]
  3. Anton L. C., Schubert U., Bacik I., Princiotta M. F., Wearsch P. A., Gibbs J., Day P. M., Realini C., Rechsteiner M. C., Bennink J. R., Yewdell J. W.. 1999; Intracellular localization of proteasomal degradation of a viral antigen. Journal of Cell Biology146:113–124
    [Google Scholar]
  4. Ben-Bassat H., Goldblum N., Mitrani S., Goldblum T., Yoffey J. M., Cohen M. M., Bentwich Z., Ramot B., Klein E., Klein G.. 1977; Establishment in continuous culture of a new type of lymphocyte from a ‘Burkitt like’ malignant lymphoma (line D G.-75). International Journal of Cancer19:27–33
    [Google Scholar]
  5. Bush K. T., Goldberg A. L., Nigam S. K.. 1997; Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance. Journal of Biological Chemistry272:9086–9092
    [Google Scholar]
  6. Chelbi-Alix M. K., de The H.. 1999; Herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated PML and Sp100 proteins. Oncogene18:935–941
    [Google Scholar]
  7. Ciechanover A.. 1998; The ubiquitin–proteasome pathway: on protein death and cell life. EMBO Journal17:7151–7160
    [Google Scholar]
  8. Cludts I., Farrell P. J.. 1998; Multiple functions within the Epstein–Barr virus EBNA-3A protein. Journal of Virology72:1862–1869
    [Google Scholar]
  9. Finke J., Rowe M., Ernberg I., Rosen A., Dillner J., Klein G.. 1987; Monoclonal and polyclonal antibodies against Epstein–Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt’s lymphoma and lymphoblastoid cell lines. Journal of Virology61:3870–3878
    [Google Scholar]
  10. Fourie A. M., Hupp T. R., Lane D. P., Sang B. C., Barbosa M. S., Sambrook J. F., Gething M. J.. 1997; HSP70 binding sites in the tumor suppressor protein p53. Journal of Biological Chemistry272:19471–19479
    [Google Scholar]
  11. Harada S., Kieff E.. 1997; Epstein–Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. Journal of Virology71:6611–6618
    [Google Scholar]
  12. Hightower L. E.. 1991; Heat shock, stress proteins, chaperones, and proteotoxicity. Cell66:191–197
    [Google Scholar]
  13. Holmvall P., Szekely L.. 1999; Computer programs that allow fast aquisition, visualization and overlap quantitation of fluorescent 3D microscopic objects by using nearest-neighbor deconvolution algorithm. Applied Immunohistochemistry & Molecular Morphology7:226–236
    [Google Scholar]
  14. Ishov A. M., Sotnikov A. G., Negorev D., Vladimirova O. V., Neff N., Kamitani T., Yeh E. T., Strauss J. F.III., Maul G. G.. 1999; PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1. Journal of Cell Biology147:221–234
    [Google Scholar]
  15. Kim D., Kim S. H., Li G. C.. 1999; Proteasome inhibitors MG132 and lactacystin hyperphosphorylate HSF1 and induce hsp70 and hsp27 expression. Biochemical and Biophysical Research Communications254:264–268
    [Google Scholar]
  16. King W., Thomas-Powell A. L., Raab-Traub N., Hawke M., Kieff E.. 1980; Epstein–Barr virus RNA. V. Viral RNA in a restringently infected, growth-transformed cell line. Journal of Virology36:506–518
    [Google Scholar]
  17. Kitay M. K., Rowe D. T.. 1996; Protein–protein interactions between Epstein–Barr virus nuclear antigen-LP and cellular gene products: binding of 70-kilodalton heat shock proteins. Virology220:91–99
    [Google Scholar]
  18. Kubbutat M. H., Jones S. N., Vousden K. H.. 1997; Regulation of p53 stability by Mdm2. Nature387:299–303
    [Google Scholar]
  19. Mannick J. B., Tong X., Hemnes A., Kieff E.. 1995; The Epstein–Barr virus nuclear antigen leader protein associates with hsp72/hsc73. Journal of Virology69:8169–8172
    [Google Scholar]
  20. Muller S., Dejean A.. 1999; Viral immediate-early proteins abrogate the modification by SUMO-1 of PML and Sp100 proteins, correlating with nuclear body disruption. Journal of Virology73:5137–5143
    [Google Scholar]
  21. Pinhasi-Kimhi O., Michalovitz D., Ben-Zeev A., Oren M.. 1986; Specific interaction between the p53 cellular tumour antigen and major heat shock proteins. Nature320:182–184
    [Google Scholar]
  22. Quignon F., De Bels F., Koken M., Feunteun J., Ameisen J. C., de The H.. 1998; PML induces a novel caspase-independent death process. Nature Genetics20:259–265
    [Google Scholar]
  23. Rickinson A. B., Kieff E.. 1996; Epstein–Barr virus. In Fields Virology pp2397–2436 Edited by Fields B. N., Knipe D. M., Howley P. M. Philadelphia: Lippincott–Raven;
    [Google Scholar]
  24. Rowe M., Rowe D. T., Gregory D., Young L. S., Farrell P. J., Rupani H., Rickinson A. B.. 1987; Differences in B cell growth phenotype reflect novel patterns of Epstein–Barr virus latent gene expression in Burkitt’s lymphoma cells. EMBO Journal6:2743–2751
    [Google Scholar]
  25. Scheer U., Hock R.. 1999; Structure and function of the nucleolus. Current Opinion in Cell Biology11:385–390
    [Google Scholar]
  26. Sinclair A. J., Palmero I., Peters G., Farrell P. J.. 1994; EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalisation of resting human B lymphocytes by Epstein–Barr virus. EMBO Journal13:3321–3328
    [Google Scholar]
  27. Szekely L., Selivanova G., Magnusson K., Klein G., Wiman K. G.. 1993; EBNA-5, an Epstein–Barr virus encoded nuclear antigen, binds to the retinoblastoma and p53 proteins. Proceedings of the National Academy of Sciences, USA90:5455–5459
    [Google Scholar]
  28. Szekely L., Jiang W.-Q., Pokrovskaja K., Wiman K. G., Klein G., Ringertz N.. 1995a; Reversible nucleolar translocation of Epstein–Barr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion. Journal of General Virology76:2423–2432
    [Google Scholar]
  29. Szekely L., Pokrovskaja K., Jiang W.-Q., Selivanova G., Löwber M., Ringertz N., Wiman K. G., Klein G.. 1995b; Resting B-cells, EBV-infected B-blasts and established lymphoblastoid cell lines differ in their Rb, p53 and EBNA-5 expression patterns. Oncogene10:1869–1874
    [Google Scholar]
  30. Szekely L., Pokrovskaja K., Jiang W. Q., de The H., Ringertz N., Klein G.. 1996; The Epstein–Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies. Journal of Virology70:2562–2568
    [Google Scholar]
  31. Wang Z. G., Ruggero D., Ronchetti S., Zhong S., Gaboli M., Rivi R., Pandolfi P. P.. 1998; PML is essential for multiple apoptotic pathways. Nature Genetics20:266–272
    [Google Scholar]
  32. Zheng P., Guo Y., Niu Q., Levy D. E., Dyck J. A., Lu S., Sheiman L. A., Liu Y.. 1998; Proto-oncogene PML controls genes devoted to MHC class I antigen presentation. Nature396:373–376
    [Google Scholar]
  33. Zhong S., Muller S., Ponchetti S., Freemont P. S., Dejean A., Pandolfi P. P.. 2000a; Role of SUMO-1-modified PML in nuclear body formation. Blood95:2748–2752
    [Google Scholar]
  34. Zhong S., Salomoni P., Pandolfi P. P.. 2000b; The transcriptional role of PML and the nuclear body. Nature Cell Biology2:85–90
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-82-2-345
Loading
/content/journal/jgv/10.1099/0022-1317-82-2-345
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error