Epstein-Barr virus (EBV)-host cell interactions: an epigenetic dialogue?

Epstein-Barr virus (EBV) is associated with diverse lymphoid and epithelial malignancies. Their molecular pathogenesis is accompanied by epigenetic alterations which are distinct for each of them. While lymphoblastoid cell lines (LCLs) derived from B cells transformed by EBV in vitro are characterized by a massive demethylation and euchromatinization of the viral and cellular genomes, the primarily malignant lymphoid tumour Burkitt’s lymphoma (BL) and the epithelial tumours nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC) are characterized by hypermethylation of a multitude of cellular tumour suppressor gene loci and of the viral genomes. In some cases, the viral latency and oncoproteins including the latent membrane proteins LMP1 and LMP2A and several nuclear antigens (EBNAs) affect the level of cellular DNA methyltansferases or interact with the histone modifying machinery. Specific molecular mechanisms of the epigenetic dialogue between virus and host cell remain to be elucidated

The MEC1 and MEC2 lines represent two CLL subclones in different stages of progression towards prolymphocytic leukemia.

The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells

Acetylated Histone H3 and H4 Mark the Upregulated LMP2A Promoter of Epstein-Barr Virus in Lymphoid Cells▿

We analyzed the levels of acetylated histones and histone H3 dimethylated on lysine 4 (H3K4me2) at the LMP2A promoter (LMP2Ap) of Epstein-Barr virus in well-characterized type I and type III lymphoid cell line pairs and additionally in the nasopharyngeal carcinoma cell line C666-1 by using chromatin immunoprecipitation. We found that enhanced levels of acetylated histones marked the upregulated LMP2Ap in lymphoid cells. In contrast, in C666-1 cells, the highly DNA-methylated, inactive LMP2Ap was also enriched in acetylated histones and H3K4me2. Our results suggest that the combinatorial effects of DNA methylation, histone acetylation, and H3K4me2 modulate the activity of LMP2Ap

Comparison of the MEC1 and MEC2 cells.

<p>(A) Expression of EBV encoded proteins EBNA-2 and LMP-1 by immunofluorescence; magnification (×100), scale bar 25 µm. Note: the MEC2 cells are larger. (B) Expression of EBNA-2 and LMP-1 by immunoblotting; positive control: CBM1-Ral-STO, negative control: Ramos. 1.5×10⁵ cells were loaded in control lanes and 5×10⁵ were loaded in MEC1 and MEC2 lanes. Note MEC2 expresses higher amount of EBNA-2. (C) Expression of Bright and BARF1 by Q-PCR. (D) FACS analysis of surface markers that are differently expressed in the 2 lines.</p

The effect of IL-21 and CD40L exposure on MEC1 and MEC2 cells.

<p>Expression of EBNA-2 and LMP-1 in IL-21 treated cells (A, B). (A) Simultaneous immunofluorescence staining of EBNA-2 (Green) and LMP-1 (Red); magnification (×100), scale bar 25 µm. Note the downregulation of EBNA-2 and upregultion of LMP-1 after IL-21 treatment. (B) Expression of EBNA-2, LMP-1 and Blimp-1 by immunoblotting; positive control: CBM1-Ral-STO, negative control: Ramos. 1.5×10⁵ cells were loaded in the control lanes and 5×10⁵ were loaded in both untreated and IL-21 treated MEC1 and MEC2 lanes. Note low expression of EBNA-2 and high expression of LMP-1 after IL-21 treatment and induction of Blimp-1 after IL-21 treatment. (C) Activity of the W and C promoters that regulate EBNA-2 expression and LMP-1 mRNA expression by Q-PCR. Note the difference in EBNA-2 regulation; the MEC2 cell uses both Wp and Cp while in MEC1 only Wp is active. (D) Expression of EBNA-2 and LMP-1 in cells exposed to CD40L. Simultaneous immunofluorescence staining; for details see (A). Note: EBNA-2 and LMP-1 are downregulated by CD40L in both lines. (E) CD40L induced modulation of surface marker by FACS analysis.</p