A site in the Epstein-Barr virus (EBV) transforming protein LMP1 that constitutively associates with the tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein TRADD to mediate NF-B and c-Jun N-terminal kinase activation is critical for long-term lymphoblastoid cell proliferation. We now find that LMP1 signaling through TRADD differs from TNFR1 signaling through TRADD. LMP1 needs only 11 amino acids to activate NF-B or synergize with TRADD in NF-B activation, while TNFR1 requires ϳ70 residues. Further, LMP1 does not require TRADD residues 294 to 312 for NF-B activation, while TNFR1 requires TRADD residues 296 to 302. LMP1 is partially blocked for NF-B activation by a TRADD mutant consisting of residues 122 to 293. Unlike TNFR1, LMP1 can interact directly with receptor-interacting protein (RIP) and stably associates with RIP in EBV-transformed lymphoblastoid cell lines. Surprisingly, LMP1 does not require RIP for NF-B activation. Despite constitutive association with TRADD or RIP, LMP1 does not induce apoptosis in EBV-negative Burkitt lymphoma or human embryonic kidney 293 cells. These results add a different perspective to the molecular interactions through which LMP1, TRADD, and RIP participate in B-lymphocyte activation and growth.Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) is essential for EBV-mediated growth transformation of resting primary human B lymphocytes into indefinitely proliferating lymphoblastoid cell lines (LCLs) (30). Genetic and biochemical evidence indicates that the signal transduction pathway through which LMP1 mediates B-lymphocyte growth transformation resembles those of activated tumor necrosis factor receptors (TNFR) (26,28,31,43). LMP1 consists of a 24-amino-acid N-terminal cytoplasmic domain, six transmembrane domains, and a 200-amino-acid C-terminal cytoplasmic tail (Fig. 1). Although no specific sequence of the N-terminal cytoplasmic domain is essential for growth transformation, the N terminus fulfills a critical structural role by tethering the first transmembrane domain to the cytoplasm (25, 30). The six transmembrane domains collectively enable LMP1 to self-aggregate in the plasma membrane similarly to a capped receptor. Aggregation of LMP1 in the plasma membrane causes two specific sites within the C-terminal cytoplasmic tail to constitutively mediate essential transforming signals through association with proteins that ordinarily mediate ligand-induced signals from activated TNFR family members (7,12,15,16,19,22,25,31,50,56). These two transformation effector sites (TES1 and TES2) are within C-terminal NF-B activating regions CTAR1 and CTAR2 (24,42). TES1/CTAR1 (residues 187 to 231 [ Fig. 1]) interacts with TNFR-associated factors (TRAFs) and is sufficient for mediating initial B-lymphocyte growth transformation (31, 43). TRAF1, TRAF2, and TRAF5 mediate NF-B activation from TES1/CTAR1 (4,10,43,49). Deletion of the TRAF interaction site results in EBV recombinants that are unable to growth transform primary B lymphocytes (26). The TES1/ ...
Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-κB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.
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