Characterization of Target Gene Regulation by the Two Epstein-Barr Virus Oncogene LMP1 Domains Essential for B-cell Transformation.

The Epstein-Barr virus oncogene latent membrane protein 1 (LMP1) is expressed by multiple EBV-associated malignancies, where it mimics CD40 signaling to activate growth and survival pathways. Two LMP1 cytoplasmic tail domains are essential for EBV-driven transformation of human B lymphocytes into immortalized lymphoblastoid cell lines (LCL), a model for EBV+ lymphomas of immunosuppressed hosts. Classic genetic studies defined two LMP1 C-terminal cytoplasmic tail regions, termed transformation essential sites (TES) 1 and 2, as critical for B-cell transformation. However, a longstanding question has remained how TES1 and TES2 non- redundantly versus jointly regulate key target genes. To gain insights, we conditionally expressed wildtype LMP1 versus LMP1 point mutants abrogated for TES1 and/or TES2 signaling in Burkitt B-cells with low basal NF-kB activity. RNAseq analyses revealed gene clusters that responded more strongly to TES1 versus TES2, that respond strongly to both, or that are oppositely regulated by TES1 and 2. Cross-comparison with EBV-transformed B-cell CRISPR/Cas9 screens identified TES1 and 2 effects on genes critical for LCL growth and survival, including BATF and IRF4. Similarly, bioinformatic analysis highlighted TES1 vs TES2 roles in regulation of genes targeted by EBV super-enhancers, which in LCLs are bound by all 5 NF-kB transcription factors. To further identify key LMP1 targets, we profiled LCL transcriptome-wide responses to CRISPR LMP1 knockout. Collectively, these studies suggest a model by which LMP1 TES1 and TES2 jointly remodel the B-cell transcriptome to support oncogenic growth and survival.