Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden1
Author for correspondence: Katja Pokrovskaja. Fax +46 8 33 04 98. e-mail katpok{at}ki.se
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Abstract |
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Introduction |
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EBNA-5 and EBNA-2 are the first virally encoded proteins expressed after B cell infection (Alfiery et al., 1991 ). They can drive gp340-activated primary B cells into the G1 phase of the cell cycle (Sinclair et al., 1994
). This suggests that EBNA-5 may play a role at the first steps of EBV transformation. EBNA-5 can co-operate with EBNA-2 in the activation of LMP-1 and Cp viral promoters (Harada & Kieff, 1997
). The co-transfection of EBNA-3 and EBNA-5 into the DG75 line showed that EBNA-3 is tethered to the nuclear matrix fraction in the presence of EBNA-5 (Cludts & Farrell, 1998
). This suggested that EBNA-5 may modify the intranuclear sorting of proteins.
After EBV infection of B cells, EBNA-5 is homogeneously distributed in the nucleus during the first 2 days. Later it accumulates in PML bodies or PODs (PML oncogenic domains; Szekely et al., 1995b , 1996
). These are distinct nuclear domains associated with the nuclear matrix. PML, in complex with SUMO-1, is required to form the POD structure (Ishov et al., 1999
; Zhong et al., 2000 a
). PODs are specifically disrupted in human acute promyelocytic leukaemia cells where PML is fused to a retinoic acid receptor alpha gene. A number of cellular proteins are localized to the PODs: SP100, INT6, CBP/p300, Hsp70, a fraction of Rb, Daxx and SUMO-1 (for review see Zhong et al., 2000b
). Herpes simplex virus type 1 infection abrogates the modification of PML by SUMO-1 (Muller & Dejean, 1999
), leading to rapid PML protein degradation (Chelbi-Alix & de The, 1999
) and disruption of the PODs. The EBV growth and transformation-associated EBNA-5 localizes to the PODs without disrupting them (Szekely et al., 1996
).
PML expression is induced by interferons. Cellular response to interferon requires normal PML function (Quignon et al., 1998 ; Wang et al., 1998
). PML was recently shown to regulate MHC expression in untransformed fibroblasts and to induce the expression of the proteins involved in antigen processing and presentation (Zheng et al., 1998
). The ubiquitinproteasome system is involved in the processing of MHC class I antigens, providing a link between the cellular degradation machinery and PML. The ubiquitinproteasome system is the major pathway of selective protein degradation in eukaryotic cells. Initially, the target proteins are conjugated to the polypeptide ubiquitin through a lysine residue on the proteins. In the second step, the ubiquitin-conjugated proteins are recognized by the 26S proteasome and degraded (for review see Ciechanover, 1998
). It is likely that nuclear POD structures are involved in this process. A protein genetically modified for rapid degradation can accumulate in the ubiquitinated form in the PODs upon proteasome inhibitor treatment. This was accompanied by the attraction of the proteasomes to the PODs, suggesting that the PODs may represent an intermediate reservoir for the ubiquitinated proteins targeted for degradation (Anton et al., 1999
).
In an attempt to understand the role of EBNA-5 targeting to the PML bodies and in proteasome-mediated protein degradation, we monitored the changes in subcellular localization and in the total EBNA-5 levels upon proteasome inhibitor treatment. We have also continued to analyse the subnuclear localization of EBNA-5 in different cell types and found that EBNA-5 targets preferentially the PODs in the LCLs and in the BLs with an LCL-like phenotype. Nucleoplasmic localization was found in other cell types. Independently of EBNA-5 localization to the PODs or to the nucleoplasm, EBNA-5 translocated to the nucleoli when the cells were treated with proteasome inhibitor MG132.
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Methods |
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Clones of HeLa, MCF-7 and SW480 lines stably expressing EBNA-5 from a pBabe-EBNA-5 construct were generated using selection with 1 µg/ml puromycin (Sigma). Proteasome inhibitor MG132 was purchased from Calbiochem, diluted in DMSO and used at concentrations of 520 µM for the 616 h treatment of cells. The control cells were incubated with the same amounts of DMSO. GFPEBNA-5 was made by cloning an EBNA-5-encoding BamHIEcoRI fragment from pBabe-EBNA-5, containing four W repeats and the unique C-terminal region, into BglIIEcoRI-cleaved pEGFP-C1 (Clontech).
Antibodies, immunostainings and Western blotting.
The following antibodies were used in this study for immunofluorescence and Western blotting: MAb D07 against p53 (DACO); MAb JF186 against EBNA-5 (Finke et al., 1987 ) and biotinylated JF186 (GibcoBRL biotinylation kit was used); MAb Ab-1 against Hsp72/73 (Oncogene Science); MAb pe2 against EBNA-2 (a gift from M. Rowe, University of Wales College of Medicine, UK); and MAb CS1-4 against LMP-1 (DACO). MAb against B23/nucleophosmin was a gift from P. K. Chan, Baylor College of Medicine, Houston, USA; rabbit serum against PML was a gift from H. de The, Institut dHematologie de lUniversite Paris VII, Paris, France. Cells were fixed in cold methanolacetone, rehydrated for 30 min, stained with primary MAb for 1 h followed by three washes in PBS, incubated with secondary FITC or Texas red-conjugated antibodies, washed three times and mounted with 80 % glycerol solution in PBS containing 2·5% 1,4-diazabicyclo-(2.2.2)octane (Sigma). Bisbenzimide (Hoechst 33258) was added at a concentration of 0·4 µg/ml to the secondary antibody for DNA staining. The double stainings for Hsp70 and EBNA-5 were done as follows: Ab-1 against Hsp70/rabbit-anti-mouse FITC-conjugated (DACO)/normal mouse serum/biotinylated JF186/Texas red-conjugated streptavidin (Vector). FITC-conjugated goat-anti-mouse and Texas red-conjugated goat-anti-rabbit (Vector) secondary antibodies were used for double EBNA-5/PML stainings.
The images were recorded on a DAS microscope Leitz DM RB with a Hamamatsu dual mode cooled CCD camera C4880. Alternatively, a fluorescence 3D microscope was used to reconstitute the image from a series of optical sections that were deblurred using the nearest neighbour algorithm with the help of the computer program TROOPER3, developed by us (Holmvall & Szekely, 1999 ). The original graphics files are available as 8 and 24 bit uncompressed TIFF images at our anonymous FTP site at ftp://130.237.124.100/E5LLL.
Total cell extracts were prepared by direct lysis in a hot Laemmli buffer. Proteins were separated on 10% SDSPAGE followed by transfer to nitrocellulose membrane (Schleicher & Schuell). Blocking and incubation with antibodies were performed in 5% milk in PBS and 0·2% Tween 20. Immunodetection using the ECL system (Amersham) was performed according to the manufacturers instructions.
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Results |
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We have established clones of SW480 (clones S2, S8, S9) and MCF-7 (clones M1, M2, M3 and M5) that ectopically express EBNA-5. The clones were double-stained for PML and EBNA-5. EBNA-5 was homogeneously distributed in the nucleoplasm, avoiding the nucleoli in all lines mentioned, and did not co-localize with PML. This is shown for MCF-7 clone M5 (Fig. 1A, lower row) and MCF-7 transiently transfected with GFPEBNA-5 (Fig. 1B
, lower row). Transiently transfected GFPEBNA-5 often accumulates in conglomerates of irregular shape, seen in one of the cells in Fig. 1(B)
, which do not co-localize with PML.
EBNA-5 translocates to the nucleoli after treatment with proteasome inhibitor MG132
Clones M1, M2 and M3 of MCF-7 expressed EBNA-5 at moderate levels and showed primarily homogeneous nuclear staining as shown in Fig. 2(A, panel a) for M1. Treatment with 20 µM of MG132 for 6 h led to a nearly complete translocation of EBNA-5 to the nucleoli (Fig. 2A
, panel c). The phase contrast fields are shown in Fig. 2(A
, panels b and d) to visualize the cells and the nucleoli. In order to prove the localization of EBNA-5 to the nucleolus, we double-stained M1 cells for EBNA-5 and nucleophosmin/B23 after MG132 treatment. Fig. 2(B)
shows that EBNA-5 is localized deep inside the nucleoli and is surrounded by the B23 protein. The pattern of B23 staining was not changed by MG132 treatment (not shown).
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Exogenously expressed EBNA-5 co-localized with Hsp70 in clone M5 in the nucleoplasm (MCF-7 transfected with EBNA-5, Fig. 4A). This shows that localization to PML bodies is not a prerequisite for EBNA-5/Hsp70 co-localization. We noticed an increase in nuclear staining of Hsp70 in the clones of MCF-7 and SW480 expressing EBNA-5, as compared to the vector-transfected cells (Fig. 4B
, panel b and not shown). This suggests that EBNA-5 tethers Hsp70 to the nucleus.
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Total protein levels were monitored by Western blotting (Fig. 4C). EBNA-5 levels did not change after MG132 treatment in IB-4, the H6 clone of HeLa expressing EBNA-5 or clone M5 of MCF-7. Even 16 h treatment did not change the EBNA-5 levels in clone M1 (Fig. 4C
, lower panel). Hsp70 levels were increased and a faster migrating band, representing the inducible Hsp72 protein, appeared in all MG132-treated cells including the EBNA-5-negative HeLa clone H4. This again demonstrates that Hsp70 is induced in response to MG132 and that this induction is independent of EBNA-5.
In order to test the effect of proteasome inhibition, we probed the same membrane with antibodies against p53, a short-lived protein degraded through the proteasome pathway. The presence of EBNA-5 did not change the levels of wild-type p53 in MCF-7 or in HeLa cells. MG132 treatment led to the accumulation of wild-type p53 in IB-4 and MCF-7 cells with the appearance of ubiquitinated forms of the protein in accordance with previously published data (Kubbutat et al., 1997 ). In HeLa cells, p53 is complexed with human papillomavirus-encoded E6 protein, which targets p53 for degradation. The inhibition of proteasome activity has led to the accumulation of p53 in both EBNA-5-expressing clone H6 and vector control H4 Hela cells.
Presence of EBNA-5 enhances translocation of mutant p53 to the nucleoli of SW480 cells upon treatment with proteasome inhibitor
The colorectal cancer line SW480 expresses endogenous mutant p53 at high levels. p53 is localized to the nuclei, mainly avoiding the nucleoli (Fig. 5A, panels c and e). P2 and S2 are pBabe vector- and pBabe-EBNA-5-transfected derivatives of SW480. The presence of EBNA-5 did not change the levels of mutant p53 (compare Fig. 5A
, panels c and e). Treatment with 20 µM of MG132 for 6 h led to a nearly complete translocation of EBNA-5 to the nucleoli of S2 (Fig. 5A
, panels a and b), to a partial accumulation of mutant p53 in the nucleoli in 10% of the P2 cells (Fig. 5A
, panel f) and to a significant p53 accumulation in the nucleoli in 90% of S2 cells (Fig. 5A
, panel d). Fig. 5(B)
shows p53 inside the nucleoli in the MG132-treated S2 cells at high magnification. In order to substantiate this finding, we double-stained MG132-treated S2 cells for EBNA-5 and p53. Fig. 5(C)
shows that p53 is preferentially accumulated in the nucleoli of cells when EBNA-5 is also in the nucleoli (arrows). In contrast, cells without EBNA-5 (square sign) or those where EBNA-5 has not changed its localization (asterisks) showed homogeneous p53 staining. These results show that EBNA-5 greatly enhances accumulation of mutant p53 in the nucleoli upon inhibition of proteasome activity.
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Discussion |
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The Hsp70 proteins are involved in protein folding, protein translocation across membranes and thermal tolerance. In non-stressed cells, Hsp70 associates transiently with nascent polypeptide chains, polypeptides unfolded for translocation or other aberrantly folded proteins (for review see Hightower, 1991 ). Both major members of the Hsp70 family, the stress-inducible Hsp72 and the constitutive cognate Hsc73, were found to associate with the soluble form of EBNA-5 in co-immunoprecipitation experiments. The W1W2-repeats in the EBNA-5 protein were required for binding (Mannick et al., 1995
). Another study showed that the Y2 C-terminal exon region of EBNA-5 formed complexes with Hsp70 (Kitay & Rowe, 1996
). We have found previously that EBNA-5 is co-localized with Hsp70 in PODs in the LCL IB-4 under normal conditions and in the nucleoli upon heat shock (Szekely et al., 1995a
). Both Hsp72 and Hsc73 accumulate in the nucleoli in heat-shocked mammalian cells (for review see Hightower, 1991
). We found that Hsp70 localized to the nucleoli upon MG132 treatment, independently of the presence or absence of EBNA-5. Considering the direct binding of EBNA-5 to Hsp70, it is possible that the latter transports EBNA-5 into the nucleoli. On the other hand, Hsp70 accumulated in the nucleoplasm of the EBNA-5-, but not the vector control-transfected clones of MCF-7 and SW480 (Fig. 4A
and not shown). It also showed a high degree of co-localization with EBNA-5 in the nucleoplasm, suggesting that EBNA-5 expression recruits Hsp70 into complex formation. The nucleolar localization of Hsp70 in proteasome inhibitor-treated cells was enhanced by the presence of EBNA-5 (Fig. 4B
), suggesting that there is an active component in the translocation of EBNA-5 to the nucleoli rather than passive transport by Hsp70.
EBNA-5 is strongly associated with the nuclear matrix (Szekely et al., 1995a ). It can also tether another EBV-encoded nuclear protein, EBNA-3, to the nuclear matrix upon co-transfection (Cludts & Farrell, 1998
). This suggests that EBNA-5 can influence the subnuclear localization of other proteins and therefore it may play a scaffolding role by forming bridges between the nuclear matrix and nucleoplasmic proteins.
We found in this study that EBNA-5 greatly enhanced the accumulation of a mutant, but not wild-type, p53 in the nucleoli upon MG132 treatment. Mutations in p53 lead to misfolding of the protein. Mutant p53 forms complexes with Hsp70 (Pinhasi-Kimhi et al., 1986 ). The high affinity binding sites for these proteins were mapped to the hydrophobic core of the central DNA binding domain, not accessible in a wild-type conformation (Fourie et al., 1997
). In the absence of EBNA-5, mutant p53 was only rarely found in the nucleoli of MG132-treated cells. Our study suggests that EBNA-5 targets mutant p53 to the nucleoli upon proteasome inhibitor treatment. GSTEBNA-5 could precipitate mutant p53 from SW480 and Namalwa cell lysates (Szekely et al., 1993
). It is possible that EBNA-5 and Hsp70 form tri-component complexes with the misfolded mutant p53 that translocate to the nucleolus much more efficiently than the Hsp70mutant p53 complex. The absence of the EBNA-5 effect on wild-type p53 distribution upon MG132 treatment supports this notion. Alternatively, EBNA-5 binds and translocates mutant p53 independently of Hsp70.
The nucleoli are special nuclear domains involved in rRNA synthesis (fibrillar compartment) and ribosome formation (granular compartment). Nucleolar proteins are involved either in rRNA synthesis and processing or in ribosome assembly (for review see Scheer & Hock, 1999 ). The involvement of the nucleoli in proteasome-mediated protein degradation has not been documented. Our data on proteasome inhibitor-induced nucleolar accumulation of Hsp70, EBNA-5 and p53 may suggest that nucleoli can be involved in proteasome-dependent protein degradation.
EBNA-5 accumulates in the PML bodies in cells with an immunoblastic phenotype but not in type I BLs or in non-B cells, suggesting the presence of EBNA-5-associated, B-blast-specific nuclear factors that target EBNA-5 into the PODs. Alternatively, it may require an additional EBV-encoded protein(s) or EBV genome in order to localize to the PODs.
It has been suggested that PML bodies are the nuclear analogues of the aggrosomes that feed proteasomes with ubiquitinated substrates (Anton et al., 1999 ). PML bodies also contain Hsp70 even in unstressed cells (Szekely et al., 1995a
), suggesting that misfolded nuclear proteins might accumulate in the PODs. It seems unlikely that EBNA-5 itself is degraded through the proteasome pathway since it does not contain lysines, which are the targets for ubiquitination. Also, our Western blot data showed no increase in EBNA-5 protein levels upon MG132 treatment in different cell lines even after 16 h of proteasomal block. It seems therefore unlikely that the PML bodies provide an intermediate reservoir for EBNA-5 on its way for degradation. On the other hand, our data suggest that POD-associated EBNA-5 might regulate the proteasome-mediated degradation of other proteins.
The PML bodies might be indirectly involved in transcription regulation by coordinating the degradation of transcription factors. Transcription co-activators such as CBP/p300, transcriptional repressors, such as Daxx and Tax, and the tumour suppressor protein Rb were detected in the PODs (for review see Zhong et al., 2000b ). EBNA-5 was found to enhance EBNA-2-mediated transactivation of EBV promoters (Harada & Kieff, 1997
). The mechanism of this co-operation is not known. Our data raise the possibility that EBNA-5 might cooperate with EBNA-2 through modifying the degradation rate of transcription regulators.
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Acknowledgments |
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References |
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Alfiery, C., Birkenbach, M. & Kieff, E.(1991). Early events in EpsteinBarr virus infection of human B lymphocytes. Virology 181, 595-608.[Medline]
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 Biology 146, 113-124.
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 Cancer 19, 27-33.
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 Chemistry 272, 9086-9092.
Chelbi-Alix, M. K. & de The, H.(1999). Herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated PML and Sp100 proteins. Oncogene 18, 935-941.[Medline]
Ciechanover, A.(1998). The ubiquitinproteasome pathway: on protein death and cell life. EMBO Journal 17, 7151-7160.
Cludts, I. & Farrell, P. J.(1998). Multiple functions within the EpsteinBarr virus EBNA-3A protein. Journal of Virology 72, 1862-1869.
Finke, J., Rowe, M., Ernberg, I., Rosen, A., Dillner, J. & Klein, G.(1987). Monoclonal and polyclonal antibodies against EpsteinBarr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitts lymphoma and lymphoblastoid cell lines. Journal of Virology 61, 3870-3878.[Medline]
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 Chemistry 272, 19471-19479.
Harada, S. & Kieff, E.(1997). EpsteinBarr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. Journal of Virology 71, 6611-6618.[Abstract]
Hightower, L. E.(1991). Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 66, 191-197.[Medline]
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 Morphology 7, 226-236.
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 Biology 147, 221-234.
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 Communications 254, 264-268.[Medline]
King, W., Thomas-Powell, A. L., Raab-Traub, N., Hawke, M. & Kieff, E.(1980). EpsteinBarr virus RNA. V. Viral RNA in a restringently infected, growth-transformed cell line. Journal of Virology 36, 506-518.[Medline]
Kitay, M. K. & Rowe, D. T.(1996). Proteinprotein interactions between EpsteinBarr virus nuclear antigen-LP and cellular gene products: binding of 70-kilodalton heat shock proteins. Virology 220, 91-99.[Medline]
Kubbutat, M. H., Jones, S. N. & Vousden, K. H.(1997). Regulation of p53 stability by Mdm2. Nature 387, 299-303.[Medline]
Mannick, J. B., Tong, X., Hemnes, A. & Kieff, E.(1995). The EpsteinBarr virus nuclear antigen leader protein associates with hsp72/hsc73. Journal of Virology 69, 8169-8172.[Abstract]
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 Virology 73, 5137-5143.
Pinhasi-Kimhi, O., Michalovitz, D., Ben-Zeev, A. & Oren, M.(1986). Specific interaction between the p53 cellular tumour antigen and major heat shock proteins. Nature 320, 182-184.[Medline]
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 Genetics 20, 259-265.[Medline]
Rickinson, A. B. & Kieff, E.(1996). EpsteinBarr virus. In Fields Virology , pp. 2397-2436. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia:LippincottRaven.
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 EpsteinBarr virus latent gene expression in Burkitts lymphoma cells. EMBO Journal 6, 2743-2751.[Abstract]
Scheer, U. & Hock, R.(1999). Structure and function of the nucleolus. Current Opinion in Cell Biology 11, 385-390.[Medline]
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 EpsteinBarr virus. EMBO Journal 13, 3321-3328.[Abstract]
Szekely, L., Selivanova, G., Magnusson, K., Klein, G. & Wiman, K. G.(1993). EBNA-5, an EpsteinBarr virus encoded nuclear antigen, binds to the retinoblastoma and p53 proteins. Proceedings of the National Academy of Sciences, USA 90, 5455-5459.[Abstract]
Szekely, L., Jiang, W.-Q., Pokrovskaja, K., Wiman, K. G., Klein, G. & Ringertz, N.(1995a). Reversible nucleolar translocation of EpsteinBarr virus-encoded EBNA-5 and hsp70 proteins after exposure to heat shock or cell density congestion. Journal of General Virology 76, 2423-2432.[Abstract]
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. Oncogene 10, 1869-1874.[Medline]
Szekely, L., Pokrovskaja, K., Jiang, W. Q., de The, H., Ringertz, N. & Klein, G.(1996). The EpsteinBarr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies. Journal of Virology 70, 2562-2568.[Abstract]
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 Genetics 20, 266-272.[Medline]
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. Nature 396, 373-376.[Medline]
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. Blood 95, 2748-2752.
Zhong, S., Salomoni, P. & Pandolfi, P. P.(2000b). The transcriptional role of PML and the nuclear body. Nature Cell Biology 2, 85-90.
Received 2 August 2000;
accepted 18 October 2000.