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

Epstein Barr virus latent membrane protein 1 (LMP1) induces NK-[kappa]B activation through transformation effector sites (TES) 1 and 2, both of which are critical for B-lymphocyte transformation. TES2 principally activates canonical NK-[kappa]B, which we confirm is NK-[kappa]B essential modifier (NEMO)-dependent and requires an intact ubiquitin binding in A20 binding inhibitor of NK-[kappa]B and NEMO (UBAN) domain. LMP1 TES2 activated NK-[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 NK-[kappa]B activation was blocked by siRNAs to TNF[alpha] 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 NK-[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 TNF[alpha] and Tax did not. Further analysis indicated that TES2 did not activate NF-[kappa]B in cells expressing the double deletion mutant [DELTA]133-224/[DELTA]372-419. These data provide further evidence of the essential role for NEMO in LMP1 TES2 NF-[kappa]B activation and highlight the importance of unique domains within NEMO for sensing distinct NF-[kappa]B stimuli. I[kappa]B kinase [gamma] | tumor necrosis factor alpha | human T-cell leukemia virus | Tax | CD40 doi/ 10.1073/pnas.1011752107