The Epstein–Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kB

The Epstein–Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxylterminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucinemutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-kB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kB activation also appears to be a critical component of long-term outgrowth. Epstein–Barr virus (EBV) infection of B lymphocytes is nonpermissive for virus replication. Instead, EBV expresses two latent membrane proteins (LMPs) and six nuclear proteins. These proteins efficiently transform resting B lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs) (reviewed in ref. 1). LMP1 is a key effector of EBV-mediated transformation. LMP1 has oncogene-like activity in rodent fibroblast cell lines (2–4). In non-EBV-infected B lymphoma cells, LMP1 activates NF-kB and induces most of the changes associated with EBV infection, including up-regulated expression of activation markers, adhesion molecules, and Bcl-2 (5–7). In epithelial cells, LMP1 causes hypertrophy and alters differentiation (8, 9). LMP1 is essential for EBV to growth-transform B lymphocytes into LCLs (10) and is expressed in most malignancies associated with EBV infection, including lymphoproliferative disease, Hodgkin disease, and nasopharyngeal carcinoma (reviewed in ref. 11). Recombinant EBV genetic and biochemical analyses are consistent with a model that LMP1 is a constitutively activated growth factor receptor that signals through its cytoplasmic carboxyl terminus (Fig. 1). The six transmembrane domains constitutively aggregate LMP1 in the plasma membrane without exogenous ligand. Aggregation is essential for B lymphocyte transformation (10, 12). The six transmembrane domains are also essential for oncogene-like activity in rodent fibroblast cell lines and phenotypic changes in B lymphoma cells (6, 13, 14). The importance of the amino and carboxyl-terminal cytoplasmic tails has been difficult to assess in rodent fibroblast transformation assays. Some assays indicate the amino terminus is important for transformation (13) whereas others implicate the carboxyl-terminal tail (4). The cytoplasmic amino terminus is not an effector of B lymphocyte transformation but is important for tethering the first transmembrane domain to the cytoplasm (12). By contrast, the cytoplasmic carboxyl terminus is essential for B lymphocyte growth transformation and therefore is implicated in signaling (15). One B lymphocyte transformation effector site (TES1) in the cytoplasmic carboxyl terminus interacts with tumor necrosis factor (TNF) receptor (TNFR) signaling pathways. The first evidence for TES1 was from EBV recombinant genetic experiments in which a mutated LMP1 MS231 was sufficient for transforming primary B lymphocytes into LCLs that required fibroblast feeder cells for long-term outgrowth. MS231 consists of the amino terminus, six transmembrane domains, and membrane proximal 45 residues of the cytoplasmic tail (15). Further studies of MS231 recombinant virus revealed that infected cells proliferate for several weeks, but thereafter most of these cells replicate progressively more slowly and then die. In contrast, nearly all wild-type EBV-transformed LCLs are immortal (K.M. Kaye, K.I., E. Johanssen, and E.K., unpublished work). TES1 was delineated as a potential signaling motif with the finding that residues within the membrane proximal 45 amino acids of the cytoplasmic tail engage TNFR-associated factors (TRAFs) (16). Deletion of the TRAF binding site results in a nontransforming EBV (17). In LCLs, LMP1 is constitutively associated with TRAF3 and TRAF1 and, to a lesser extent, with TRAF2 (18). In contrast, TRAFs are recruited to TNFR2 or to TNFR family members such as CD40 and LTb receptor in response to ligand binding and receptor aggregation (19–21). Moreover, mimicry of an activated TNFR is concordant with the similar effects of LMP1 and activated CD40 on B lymphocyte growth and gene activation