Elena Kashuba
Karolinska Institutet
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Featured researches published by Elena Kashuba.
Journal of General Virology | 1999
Laszlo Szekely; Csaba Kiss; Karin Mattsson; Elena Kashuba; Katja Pokrovskaja; A. Juhász; Pia Holmvall; George Klein
Subnuclear distribution of the human herpesvirus-8 (HHV-8)- encoded nuclear protein LNA-1 was analysed at high resolution in body cavity (BC) lymphoma-derived cell lines, in cell hybrids between BC cells and various human and mouse cells and in freshly infected K562 and ECV cell lines. Three-dimensional reconstruction of nuclei from optical sections and quantitative analysis of the distribution of LNA-1 fluorescence in relation to chromatin showed that LNA-1 associates preferentially with the border of heterochromatin in the interphase nuclei. This was further confirmed in the following systems: in endo- and exonuclease-digested nuclei, in human-mouse (BC-1-Sp2- 0) hybrids and on chromatin spreads. LNA-1 was found to bind to mitotic chromosomes at random. Epstein-Barr virus (EBV), but not HHV-8, was rapidly lost from mouse-human hybrid cells in parallel with the loss of human chromosomes. HHV-8 could persist on the residual mouse background for more than 8 months. In early human-mouse hybrids that contain a single fused nucleus, LNA-1 preferentially associates with human chromatin. After the gradual loss of the human chromosomes, LNA-1 becomes associated with the murine pericentromeric heterochromatin. In human-human hybrids derived from the fusion of the HHV-8-carrying BCBL-1 cells and the EBV-immortalized lymphoblastoid cell line IB4, LNA-1 did not co-localize with EBNA-1, EBNA-2, EBNA-5 or EBNA-6. LNA-1 was not associated with PML containing ND10 bodies either. DNase but not RNase or detergent treatment of isolated nuclei destroys LNA-1 bodies. In advanced apoptotic cells LNA- 1 bodies remain intact but are not included in the apoptotic bodies themselves.
Biochemical and Biophysical Research Communications | 2010
George Klein; Eva Klein; Elena Kashuba
Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.
Journal of General Virology | 2002
Karin Mattsson; Csaba Kiss; Georgina Platt; Guy R. Simpson; Elena Kashuba; George Klein; Thomas F. Schulz; Laszlo Szekely
LANA, the major latency-associated nuclear antigen of Kaposis sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV-8), binds RING3 protein, one of five human homologues of the fsh (female sterile homeotic) gene product of Drosophila. In KSHV/HHV-8-infected cells LANA and the viral episomes accumulate in heterochromatin-associated nuclear bodies. Here we show that in several KSHV/HHV-8-negative cell lines derived from carcinomas, sarcomas and lymphomas, RING3 was expressed at low levels, primarily localized to the euchromatin, and dissociated from the chromosomes during mitosis. In contrast, in KSHV/HHV-8-infected body cavity lymphoma cells the bulk of RING3 localizes to the LANA nuclear bodies and remains associated with the chromosomes during cell division. KSHV/HHV-8-infected body cavity lymphoma cells expressed RING3 at much higher levels than cells without the virus. Transfection of full-length LANA, but not the C terminus alone, greatly induced RING3 gene expression, and LANA and RING3 co-localized even in the transfected cells, in the absence of KSHV/HHV-8 viral DNA. High levels of LANA expression led to the disappearance of heterochromatin in both human and mouse cells. We suggest that LANA and RING3 may create a local euchromatic microenvironment around the viral episomes that are anchored to the heterochromatin.
Oncogene | 2000
Elena Kashuba; Katja Pokrovskaja; George Klein; Laszlo Szekely
EBNA-3 (also called EBNA-3A) is one of the EBV encoded nuclear antigens that are necessary for B-cell transformation. EBNA-3 is known to target RBPs, nuclear proteins that also interacts with EBNA-2, EBNA-4 and EBNA-6. In order to identify additional EBNA-3 targets, an EBV-transformed human lymphocyte cDNA library was screened in the yeast two-hybrid system with N-terminus truncated EBNA-3 that cannot interact with RBP-Jκ. A clone, encoding Xap-2 protein, a cellular partner of Hepatitis B virus X-antigen was isolated. This protein is also known as the p38 subunit of the aryl hydrocarbon receptor complex (ARA9). The specific binding to EBNA-3 was confirmed by showing that the GST-Xap-2 precipitated EBNA-3 from CV1 cells that were infected with recombinant vaccinia virus expressing EBNA-3. Deletion of the C-terminus of Xap-2 eliminated the binding. Fusion with green fluorescent protein showed that Xap-2 is preferentially cytoplasmic but translocates to the nucleus upon expression of EBNA-3.
Journal of Biological Chemistry | 2006
Elena Kashuba; Katarina Gradin; Marja Isaguliants; Laszlo Szekely; Lorenz Poellinger; George Klein; Arunas Kazlauskas
EBNA-3 is one of the Epstein-Barr virus (EBV)-encoded nuclear antigens that is indispensable for immunoblastic transformation and sustained proliferation of B-lymphocytes. The molecular mechanisms responsible for the function of EBNA-3 are poorly understood. We previously found that EBNA-3 interacts with an immunophilin-like protein XAP2/ARA9/AIP, which in mammalian cells is known to interact with the latent aryl hydrocarbon receptor (AhR). AhR is a ligand-inducible transcription factor that mediates cellular responses to environmental pollutants, such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD). In this study, we show that EBNA-3 interacts specifically with AhR. The stability of this interaction is determined by the activation state of AhR and its association with XAP2. We and others have demonstrated that XAP2 retains the nonactivated AhR in the cell cytoplasm. However, in the presence of TCDD, the effect of XAP2 on the intracellular localization of AhR was counter-acted by EBNA-3, resulting in nuclear translocation of the AhR. In addition, EBNA-3 enhanced transactivation function by the ligand-activated AhR in cells, as assessed by reporter gene assays. Our data suggested that EBNA-3 plays a role in facilitating the ligand-dependent AhR activation process. Following activation of the AhR, we also observed that EBNA-3 counteracted the inhibitory effect of TCDD on the growth of EBV-carrying lymphoblasts. Taken together, our studies revealed a novel interaction between EBV- and AhR-dependent cellular pathways that control cell proliferation and survival.
PLOS ONE | 2012
Suhas Darekar; Konstantinos Georgiou; Mariya Yurchenko; Surya Pavan Yenamandra; Georgia Chachami; George Simos; George Klein; Elena Kashuba
Background Epstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied. Methods and Findings Using Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate. Conclusions Our data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.
Journal of General Virology | 2001
Katja Pokrovskaja; Karin Mattsson; Elena Kashuba; George Klein; Laszlo Szekely
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.
Proceedings of the National Academy of Sciences of the United States of America | 2008
Elena Kashuba; Mariya Yurchenko; Surya Pavan Yenamandra; Boris Snopok; Maria G. Isaguliants; Laszlo Szekely; George Klein
Epstein–Barr virus (EBV), like other DNA tumor viruses, induces an S-phase in the natural host cell, the human B lymphocyte. This is linked with blast transformation. It is believed that the EBV-encoded nuclear antigen 6 (EBNA-6) is involved in the regulation of cell cycle entry. However, the possible mechanism of this regulation is not approached. In our current study, we found that EBNA-6 binds to a MRPS18-2 protein, and targets it to the nucleus. We found that MRPS18-2 binds to both hypo- and hyperphosphorylated forms of Rb protein specifically. This binding targets the small pocket of pRb, which is a site of interaction with E2F1. The MRPS18-2 competes with the binding of E2F1 to pRb, thereby raising the level of free E2F1. Our experimental data suggest that EBNA-6 may play a major role in the entry of EBV infected B cells into the S phase by binding to and raising the level of nuclear MRPS18-2, protein. This would inhibit pRb binding to E2F1 competitively and lift the block preventing S-phase entry.
International Journal of Cancer | 2003
Elena Kashuba; Karin Mattsson; Katja Pokrovskaja; Csaba Kiss; Marina Protopopova; Barbro Ehlin-Henriksson; George Klein; Laszlo Szekely
Epstein‐Barr virus (EBV) carrying lymphoblastoid cells of normal origin express the full program of all 9 virus‐encoded, growth transformation associated proteins. They have an intact p53 pathway as a rule. This raises the question of whether any of the viral proteins impair the pathway functionally. Using a yeast 2‐hybrid system, we have shown that EBNA‐5 but not the other EBNAs interacts with the p14ARF protein, a regulator of the p53 pathway. The interaction was confirmed in vitro using a GST pull‐down assay. Moreover, expression of EBNA‐5 increased the survival of p14ARF‐transfected cells. EBV infection of resting B cells induced the expression of p14ARF mRNA without increased level of the protein. A fraction of the p14ARF localized to the nucleoli but the bulk of the protein accumulated in nuclear but extranucleolar inclusions. Formation of the extranucleolar inclusions led to complete relocalization of EBNA‐5 from nucleoplasm to these structures. The inclusions also contained p53 and HDM2, and were surrounded by PML bodies and proteasomes, which suggests that these inclusions could be targets for proteasome dependent protein degradation.
Cellular and Molecular Life Sciences | 2010
Surya Pavan Yenamandra; Ulf Hellman; Bettina Kempkes; Suhas Darekar; Sabine Petermann; Tom Sculley; George Klein; Elena Kashuba
Epstein-Barr virus (EBV) is a human gamma herpes virus that infects B cells and induces their transformation into immortalized lymphoblasts that can grow as cell lines (LCLs) in vitro. EBNA-3 is a member of the EBNA-3-protein family that can regulate transcription of cellular and viral genes. The identification of EBNA-3 cellular partners and a study of its influence on cellular pathways are important for understanding the transforming action of the virus. In this work, we have identified the vitamin D receptor (VDR) protein as a binding partner of EBNA-3. We found that EBNA3 blocks the activation of VDR-dependent genes and protects LCLs against vitamin-D3-induced growth arrest and/or apoptosis. The presented data shed some light on the anti-apoptotic EBV program and the role of the EBNA-3-VDR interaction in the viral strategy.