The role of EBNA binding proteins in cell transformation

Epstein-Barr virus (EBV) infects majority of the human population and maintains sub-clinical infection. However, under certain conditions it is associated with several B-cell malignancies, such as Burkitt lymphoma, Hodgkin’s lymphoma etc. Moreover, EBV also plays a causative role in acquired immunodeficiency syndrome (AIDS) associated lymphomas and post-transplant lymphoproliferative disease (PTLD). EBV maintains latent infection and expresses a particular set of proteins that are necessary for host cell proliferation. Studying function of EBV latent proteins could help us to understand the mechanisms underlying EBV induced B-cell transformation.

EBV transformed B cells, i.e. lymphoblastoid cell lines (LCLs) is a well- established in vitro model system to study the molecular mechanisms of B-cell transformation. In the present work, we have identified vitamin D receptor (VDR) as a binding partner of EBNA3. We showed that EBNA3 can block the VDR mediated gene transactivation and protects B-cells from vitamin D3 induced growth arrest/ apoptosis. We have observed that hypoxia inducible factor 1 alpha (HIF1α) is stabilized in LCLs at normoxic conditions. HIF1α is not hydroxylated and therefore it is not degraded in LCLs. We have shown that prolylhydroxylases 1 and 2 (PHD1 and 2) that are responsible for hydroxylation of HIF1α, form complexes with EBNA5 and EBNA3, respectively. Due to this binding catalytic activity of PHDs is blocked, resulting in inhibition of HIF1α hydroxylation and subsequent degradation. Stabilized HIF1α is transcriptionally active and induces genes that are involved in glycolysis. Moreover, LCLs have high levels of pyruvate and lactate in contrast to mitogen activated B cells, indicating induction of aerobic glycolysis or Warburg effect.

We have shown that mitochondrial ribosomal protein MRPS18-2 (S18-2), an EBNA6 binding protein, can immortalize rat embryonic fibroblasts (REFs). These immortalized cells express stem cell markers like SSEA1, Sox2, Oct3/4 and have the characteristics of embryonic stem cells. S18-2 also immortalized the adult rat skin fibroblasts (RSFs). Moreover, single clones from immortalized REFs and RSFs resulted in tumors in SCID mice. This thesis work reveals three different aspects of EBV induced B-cell transformation, i.e. protection from vitamin D3 induced apoptosis, metabolic adaptation required for proliferation and hijacking functions of novel protein MRPS18-2 for immortalization.

List of scientific papers

I. Yenamandra SP, Hellman U, Kempkes B, Darekar SD, Petermann S, Sculley T, Klein G, Kashuba E. (2010). Epstein-Barr virus encoded EBNA-3 binds to vitamin D receptor and blocks activation of its target genes. Cell Mol Life Sci. 2010. 67(24): p. 4249-56.
https://doi.org/10.1007/s00018-010-0441-4

II. Darekar SD, Georgiou K, Yurchenko M, Yenamandra SP, Chachami G, Simos G, Klein G, Kashuba E. (2012). Epstein-Barr virus immortalization of human B-cells leads to stabilization of hypoxia-induced factor 1 alpha, congruent with the Warburg effect. PLoS One. 2012. 7(7): p. e42072.
https://doi.org/10.1371/journal.pone.0042072

III. Kashuba E, Pavan Yenamandra S, Darekar SD, Yurchenko M, Kashuba V, Klein G, Szekely L. (2009). MRPS18-2 protein immortalizes primary rat embryonic fibroblasts and endows them with stem cell-like properties. Proc Natl Acad Sci U S A. 2009. 106(47): p. 19866-71.
https://doi.org/10.1073/pnas.0911545106

IV. Yenamandra SP, Darekar SD, Kashuba V, Matskova L, Klein G, Kashuba E. (2012). Stem cell gene expression in MRPS18-2-immortalized rat embryonic fibroblasts. Cell Death Dis. 2012. 3: p. e357.
https://doi.org/10.1038/cddis.2011.138

V. Darekar SD, Gurrapu S, Drummond C, Matskova L, Kashuba E. Overexpression of the mitochondrial ribosomal protein S18-2 results in transformation of primary rat skin fibroblasts. [Manuscript]