The Epstein-Barr Virus (EBV) Encoded Oncoprotein LMP1 Mediates Down Regulation of Shelterin Proteins, Formation of Telomere Aggregates and Multinuclearity.

Tumour cells in classical Hodgkin's lymphoma (HL) and Burkitt's lymphoma (BL) are derived from germinal center B-cells and may or may not harbour the EBV genome. In both, EBV-negative and EBV-associated HL, the 3D nuclear organization of telomeres marks the transition from mononuclear Hodgkin (H-cell) to diagnostic multinuclear Reed-Sternberg (RS-cell) cells. Moreover, transient expression of the EBV-encoded oncoprotein LMP1 in the HL cell lines L-428 and HD-MyZ significantly increases the percentage of RS cells. However, the molecular mechanisms through which this selective LMP1 oncoprotein expression promotes the transition from H- to RS-cells is still unknown. In order to answer this question we analyzed the 3D telomere dynamics and the expression of key shelterin proteins at the transcriptional, translational and topographical localization level in the EBV-negative Burkitt cell line BJAB, stably transfected with an inducible LMP1 system (tetracycline on/off system). To mimic the permanent LMP1 expression as observed in tumour cells of EBV-associated Hodgkin's lymphoma, we extended the period of LMP1 expression up to 21 days and examined key proteins of the shelterin complex, 3D telomere dynamics and formation of multinuclear cells in interphase nuclei of BJAB Burkitt's lymphoma cells at day 1, 3, 7, 10, 14 and 21. Statistical analysis was performed using nested or two-way analysis of variance.

Already at day 3 LMP1 induced substantial down regulation of the shelterin key-components TRF1, TRF2 and POT1 at the transcriptional and protein level. This down regulation was reversible after LMP1 suppression; in particular, suppression of LMP1 induction at day 7 returned shelterin-key components to the initial level of expression at day 14. Massive LMP1 mediated down regulation of shelterin proteins was also confirmed by immunofluorescence. At day 7, stable LMP1 expression led to a significant increase of 3D telomere aggregates, nuclear volume and polyploid cells, most of them multinucleated, when compared to non-LMP1 expressing BJAB controls (p < 0.02). These changes progressed over time and at day 21 LMP1 expressing BJAB cells were characterized by highly significant increase of 3D telomere aggregates (p = 0.0005) and increased nuclear volume (p < 0.0001), compared to the LMP1-suppressed control. Discussion: In an experimental setting mimicking the in vivo conditions of EBV-associated HL, i.e. a permanent LMP1 expression in the H-cells, the LMP1 oncoprotein deregulates shelterin-expression, leads to telomere aggregates and mediates multinuclearity. Our findings point to a shelterin/telomere related key function of LMP1 in Reed-Sternberg cell formation in EBV-associated Hodgkin's lymphoma and may explain the four fold increased risk to develop HL within a median incubation time of four years after symptomatic EBV infection. The LMP1-dependent mechanisms involved are probably unprotected telomeres (shelterin down regulation) favouring chromosomal rearrangements as a result of telomeric aggregate formation and ongoing bridge-fusion- bridge cycles which lead to disturbed cytokinesis and finally multinuclearity.

No relevant conflicts of interest to declare.