5. Bidisha Mitra, PhD

Epstein-Barr virus (EBV) causes B-cell lymphomas, including post-transplant lymphoproliferative disorder (PTLD). The EBV oncogene Latent membrane protein 1 (LMP1) is required for EBV conversion of primary human B cells into immortalized lymphoblastoid cell lines, a major PTLD model. Reverse genetics studies identified that two LMP1 C-terminal regions, termed Transformation Effector sites (TES) 1 and 2, are each necessary for B-cell immortalization. However, a longstanding question has remained how TES1 and 2 function non-redundantly in EBV-mediated B-cell transformation. Our central hypothesis is that TES1 is required for transformation initiation and TES2 is required to maintain the transformed B-cells.
We have used the EBV bacterial artificial chromosome system to engineer point mutations in TES1 and/or TES2 that abrogate signaling and generate EBV virus. We observed that primary human B-cells when infected with EBV carrying wildtype, TES1 mutant (TES1m), TES2 mutant (TES2m) or TES1/2 double mutant (DM) LMP1, the TES1m and DM fail to survive 10 days post infection. We have also identified that the observed cell death is due to TNF-alpha mediated extrinsic apoptosis and can be rescued through chemical inhibition of Caspase 8 activity. RNAseq analysis of the infected primary B cells helped identify CFLAR that encodes cellular FLICE-inhibitory protein (c-FLIP), a master anti- apoptotic regulator, severely downregulated by the TES1m but not by TES2m. Secondly, we also recorded that the TES2m infected primary B cells undergo cell death and growth arrest at Day 21 post infection. Our preliminary data indicate abrogated glutathione metabolism and currently studies are underway to tease the mechanism out. Collectively, our study identifies non-redundant key roles played by and defined key time points when each of the TES domains of LMP1 in B-cell immortalization giving us insights into one of the oldest question in the EBV mediated B-cell transformation arena.