Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr Virus LMP1 Domain Important for B-Lymphocyte Transformation: Role in NF-κB Activation

Abstract

The Epstein-Barr virus (EBV) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor (TNFR) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus (CT) with TNFR-associated factors (TRAFs). We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5% of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3. TRAF1, TRAF2, and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids (aa) 199 to 214, within a domain which is important for B-lymphocyte growth transformation (aa 187 to 231). Latent-infection membrane protein 1 (LMP1) of EpsteinBarr virus (EBV) is essential for the ability of EBV to induce continuous proliferation of primary human B lymphocytes (20). LMP1 comprises an N-terminal cytoplasmic domain (amino acids [aa] 1 to 24), six markedly hydrophobic transmembrane domains separated by short reverse turns (aa 25 to 186), and a long cytoplasmic carboxyl terminus (CT) (aa 187 to 386) (6) (Fig. 1). Recombinant EBV genetic analyses indicate that the six transmembrane domains and the CT first 45 aa are sufficient for primary B-lymphocyte growth transformation in the context of a specifically mutated EBV recombinant (18,20,21,23).Several lines of evidence indicate that LMP1 is a constitutively activated tumor necrosis factor family receptor (TNFR) similar to activated CD40. First, expression of LMP1 in B lymphoblasts or in primary B lymphocytes results in NF-B activation and expression of Bcl-2, activation markers, adhesion molecules, and autocrine growth factors, all of which are induced in normal B lymphocytes after stimulation by 11,16,24,26,30,33,41,42). Second, a significant fraction of LMP1 constitutively aggregates in a patch in the plasma membrane of lymphoblastoid cell lines (LCLs) (12,26,27,29). The aggregation is dependent on the six hydrophobic transmembrane domains and is essential for transformation. Thus, LMP1 comprising only the last five transmembrane domains and the entire CT, i.e., LMP1 aa 44 to 386, diffusely distributes in the plasma membrane and is nontransforming (20, 26), whereas LMP1 comprising the last two transmembrane domains and the CT, i.e., LMP1 aa 129 to 386, diffusely distributes in all cytoplasmic membranes and has no effect on lymphoblasts (16,26,41). Third, the LMP1 CT aa 187 to 231, which are sufficient for transformation when linked to the six transmembrane domains [LMP1(1-231)] (23), interact with a protein that also interacts with the CD40 cytoplasmic domain (3,15,31,39); a CD40 cytoplasmic domain nonsignaling mutant fails to interact with this protein (15). This protein (previously called LAP1, CD40bp, CRAF1, or CAP1) is now designated TRAF3 because of its extensive C-terminal "TRAF domain" homology to TNFR-associated factor 2 (TRAF2) and TRAF1 (also called EBI6) (36, 37). The LMP1 CT can also bind to TRAF2 in vitro and associates with TRAF1, TRAF2, and TRAF3 in the plasma membrane of tran...

Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth

“…In response to HGF, the intracellular portion of the c-Met proto-oncogene receptor becomes autophosphorylated and binds to a number of proteins, including phosphatidylinositol 3-kinase, phospholipase, Cg, pp60 c-src , Grb-2, SHC, and GAB1 (Bardelli et al, 1992;Ponzetto et al, 1994;Pelicci et al, 1995). The CTAR1 domain of LMP1, which was reported to induce expression of EGFR through the TRAF signaling pathway Devergne et al, 1996;Kaye et al, 1996), was responsible for transformation of MDCK cells. However, the CTAR2 domain, which is the dominant NF-kB and JNK activating region (Huen et al, 1995;Kaye et al, 1995;Eliopoulos and Young, 1998), was not essential for transformation of MDCK cells.…”

Epstein-Barr virus transformation: involvement of latent membrane protein 1-mediated activation of NF-κB

“…Recombinant viral genetic and biochemical analyses show that these two sites in the CTD of LMP1 are critical for growth transformation and activation of NF-kB (Huen et al, 1995;Kaye et al, 1995;Mitchell and Sugden, 1995). The ®rst region spans residues 187 ± 231 and constitutively interacts with the tumor necrosis factor (TNF) receptor associated factors TRAF1, TRAF2, TRAF3 and TRAF5 (Brodeur et al, 1997;Devergne et al, 1996Devergne et al, , 1998Mosialos et al, 1995). The second region of the CTD encompasses residues 352 ± 386 and this region continuously interacts with TNF receptor-associated death domain-containing protein (TRADD) and TNF receptor-interacting protein (RIP) (Eliopoulos et al, 1999a;Izumi et al, 1999; (Figure 1).…”

Expression of the Tumor Necrosis Factor Receptor-Associated Factors (TRAFs) 1 and 2 is a Characteristic Feature of Hodgkin and Reed-Sternberg Cells

“…In mammalian cells, the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family consists of a group of six adapter proteins (TRAF1-TRAF6) that participate in the intracellular signaling activity of several members of the TNF receptor (TNFR) superfamily, including the TNF receptor 2 (TNF-R2), CD30, CD40, the lymphotoxin-␤ receptor, and the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1; [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Structurally, TRAF proteins are composed of an N-terminal cysteine/histidine-rich region containing zinc RING and/or zinc finger motifs, a central coiled coil region, and a C-terminal homology region that defines the TRAF family (1,2,10,11,13).…”

“…Furthermore, in a significant portion of HD cases, HRS cells express the EBV protein LMP1 (32,33). LMP1 also contains a TRAF-binding domain and uses TRAF proteins in some aspects of its intracellular signaling (8,9,32,33).…”

“…Previous investigations have shown that in several cell types, overexpression of TRAFs 1, 2, 5, and 6 can induce activation of NF-B, resulting in both cytokine secretion and resistance to apoptosis (4 -10, 14, 15, 36). In contrast, overexpression of TRAF3 has been shown to inhibit NF-B signaling (5,9). Investigation of TRAF protein expression in HRS cells, however, has been limited and has had somewhat heterogeneous results (37,38).…”