The Epstein-Barr Virus Oncoprotein Latent Membrane Protein 1 Engages the Tumor Necrosis Factor Receptor-Associated Proteins TRADD and Receptor-Interacting Protein (RIP) but Does Not Induce Apoptosis or Require RIP for NF-κB Activation

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. ​(Fig.1).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-κB activating regions CTAR1 and CTAR2 (24, 42). FIG. 1 Diagram of LMP1. The Flag epitope was introduced at the amino terminus (NH2). LMP1 residues 187, 231, 352, and 386 are marked. LMP1 constitutively aggregates in the plasma membrane and associates with TRAFs, TRADD, and RIP. TES1 aggregates TRAFs to mediate ... 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-κB 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/CTAR1 amino acid sequence is similar to sites in CD40 and CD30 that are critical for NF-κB activation (9, 40). CD40 is the receptor for a T-cell surface ligand that activates B-lymphocyte growth and differentiation (2, 8, 32, 44), and CD30 is the receptor for a T-cell surface ligand that can negatively affect heavy-chain class switching (5, 36). In retrospect, LMP1 mimicry of an activated TNFR is not surprising given the similar effects of LMP1 and CD40 on B-lymphocyte growth and gene activation (2, 8, 9, 23, 29, 32, 44, 47). TES2/CTAR2 is at the LMP1 C terminus and activates NF-κB through the TNFR1-associated death domain protein TRADD (28). The adverse consequences of mutation of the three C-terminal LMP1 codons from YYD to ID on TRADD interaction in the yeast Saccharomyces cerevisiae, on TRADD association in mammalian cells, on NF-κB activation, on TRADD synergy with LMP1 in NF-κB activation, and on B-lymphocyte growth transformation link TRADD to these TES2/CTAR2 effects (28). The tyrosines are not specifically required since substitution with codons encoding phenylalanine is indistinguishable from wild type in TRADD association in lymphocytes, in NF-κB activation, and in B-lymphocyte growth transformation (28). Thus, at this level of genetic analysis, TES2 appears to coincide with CTAR2 (15), and TRADD is a key signal-transducing intermediate. The constitutive association of LMP1 with TRADD in LCLs (28) contrasts with TNFR1, which recruits TRADD in response to ligand binding and receptor aggregation (22, 21). The objective of the experiments reported here was to investigate further the biochemistry of LMP1 TES2/CTAR2 interaction with TRADD and the expected proapoptotic effects. Since receptor-interacting protein (RIP) is implicated in TNFR1 and TRADD-mediated activation of NF-κB and apoptosis (20, 33, 51, 54), we also investigated the role of RIP in TES2/CTAR2-mediated signaling.