Epstein-Barr latent membrane protein 1 transformation site 2 activates NF-κB in the absence of NF-κB essential modifier residues 133-224 or 373-419

Epstein Barr virus latent membrane protein 1 (LMP1) induces NF-kappa B activation through transformation effector sites (TES) 1 and 2, both of which are critical for B-lymphocyte transformation. TES2 principally activates canonical NF-kappa B, which we confirm is NF-kappa B essential modifier (NEMO)-dependent and requires an intact ubiquitin binding in A20 binding inhibitor of NF-kappa B and NEMO (UBAN) domain. LMP1 TES2 activated NF-kappa B in Jurkat cell lines harboring NEMO truncated at 372 (A45) or NEMO with an in-frame deletion of 133-224 (2C), whereas TNF alpha, 12-O-Tetradecanoylphorbol-13-acetate, human T-cell leukemia virus 1 Tax, and CD40 did not. In both A45 and 2C Jurkat cell lines, LMP1 TES2-mediated NF-kappa B activation was blocked by siRNAs to TNFa receptor-associated factor 6 and NEMO, by I kappa B kinase inhibitors, and by the I kappa B alpha superrepressor, indicating that the NEMO mutants function to support canonical NF-kappa B activation. Expression of A45 or 2C mutants in NEMO-deficient murine embryonic fibroblasts reproduced the Jurkat phenotypes: LMP1 TES2 activated NF-kappa B in fibroblasts lacking NEMO amino acids 133-224 or 373-419, but TNFa and Tax didnot. Further analysis indicated that TES2didnot activateNF-kappa B in cells expressing the double deletion mutant.133-224/.372-419. These data provide further evidence of the essential role forNEMOin LMP1 TES2 NF-kappa B activation and highlight the importance of unique domains within NEMO for sensing distinct NF-kappa B stimuli.