Epstein–Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-kB through a pathway that includes the NF-kB-inducing kinase and the IkB kinases IKKa and IKKb

The Epstein–Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively aggregated pseudo-tumor necrosis factor receptor (TNFR) that activates transcription factor NF-kB through two sites in its C-terminal cytoplasmic domain. One site is similar to activated TNFRII in associating with TNFR-associated factors TRAF1 and TRAF2, and the second site is similar to TNFRI in associating with the TNFRI death domain interacting protein TRADD. TNFRI has been recently shown to activate NF-kB through association with TRADD, RIP, and TRAF2; activation of the NF-kB-inducing kinase (NIK); activation of the IkBa kinases (IKKa and IKKb); and phosphorylation of IkBa. IkBa phosphorylation on Ser-32 and Ser-36 is followed by its degradation and NF-kB activation. In this report, we show that NF-kB activation by LMP1 or by each of its effector sites is mediated by a pathway that includes NIK, IKKa, and IKKb. Dominant negative mutants of NIK, IKKa, or IKKb substantially inhibited NF-kB activation by LMP1 or by each of its effector sites. The Epstein–Barr virus (EBV) latent infection membrane protein 1 (LMP1) is a key effector of EBV-mediated effects on cell growth. Genetic analyses with EBV recombinants indicate that LMP1 is essential for the conversion of resting human B lymphocytes into lymphoblastoid cell lines (LCLs) (1). In non-EBV-infected Burkitt’s tumor lymphoblasts, LMP1 can induce most of the phenotypic changes associated with EBV transformation of resting human B lymphocytes including induction of adhesion molecule expression, activation of NFkB, up-regulation of Bcl-2, and activation of stress-activated protein kinase (for review, see refs. 2–8). LMP1 can also transform immortalized rodent fibroblast cell lines to loss of contact inhibition, lower serum dependence, anchorage independence, and tumorigenicity in nude mice (9–11). LMP1 is likely to alter cell growth by mimicking a constitutively activated tumor necrosis factor receptor (TNFR) similar to TNFRII and TNFRI, CD40, or CD30 (12–14). LMP1 has a short 24-amino acid cytoplasmic N terminus, six transmembrane domains connected by short turns, and a 200-amino acid cytoplasmic C terminus (CCT) (ref. 2 and Fig. 1). The cytoplasmic N terminus anchors the first transmembrane domain, and deletion of any specific codon does not ablate the ability of the mutated EBV recombinant to transform B lymphocytes into LCLs (15). The transmembrane domains enable LMP1 to aggregate in the plasma membrane. Aggregation in the plasma membrane is essential for activity because EBV recombinants that express a nonaggregating mutant LMP1 that diffusely distributes in the plasma membranes are unable to convert B lymphocytes into LCLs (1). Another nonaggregating mutant that diffusely distributes in all cytoplasmic membranes does not induce phenotypic changes in B lymphocytes or rodent fibroblasts or induce NF-kB (2, 4–6, 10, 11, 16). The LMP1 CCT is also essential for the transformation of resting B lymphocytes into LCLs (17). An EBV recombinant that expresses only the N terminus, the transmembrane domains, and the first 45 amino acids of the CCT can initiate B cell growth transformation and some of the infected cells can grow into long-term LCLs (17). These first 45 amino acids of the CCT include a site that can bind TNFR-associated factors (TRAFs) (12, 13, 18). These same 45 amino acids mediate a minor component of the LMP1-induced NF-kB activation and have been referred to as the C-terminal activation region 1 (CTAR1) (4, 5). A 27-codon deletion in LMP1 that includes this TRAF-binding site renders EBV recombinants incompetent for B lymphocyte growth transformation, consistent with the hypothesis that the TRAF binding site is a key transformation effector site (TES1; ref. 19). A second transformation effector site (TES2) maps to the last few residues of the CCT (14). TES2 associates with the TNFR1 death domain interacting protein TRADD (14) and correlates with the major NF-kB-activating domain (4, 5, 14, 20). TES1 and TES2 are within the C-terminal NF-kB activation regions 1 and 2 (CTAR1 and CTAR2) that mediate 30% and 70%, respectively, of LMP1-induced NF-kB activation (5, 12, 16). Binding of TRAF1 and TRAF2 to TES1 mediates NF-kB activation from CTAR1, whereas TRADD binding to TES2 mediates NF-kB activation from CTAR2 (13, 14). The genetic linkage of LMP1 effector sites for cell growth transformation with effector sites for NF-kB activation and the key role of NF-kB in lymphocyte activation (21) are compatible with the working hypotheses that TRAFs and TRADD are key mediators of LMP1 effects in cell growth transformation and that NF-kB is an important component of those effects. Pursuant to the linkage of NF-kB activation with LMP1 effects on B lymphocyte growth, we now further investigate the molecular mechanism through which LMP1 mediates NF-kB activation. TNF appears to induce NF-kB activation through a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9510106-6$2.00y0 PNAS is available online at www.pnas.org. Abbreviations: TNFR, tumor necrosis factor receptor; TRAF, TNFRassociated factor; TRADD, TNFR1 death domain interacting protein; NIK, NF-kB-inducing kinase; IKK, 1kB kinase; EBV, Epstein–Barr virus; LMP1, latent infection membrane protein 1; LCL, lymphoblastoid cell line; CCT, cytoplasmic C terminus; CTAR, C-terminal activation region; TES, transformation effector site; EST, expressed sequence tag. §To whom reprint requests should be addressed. e-mail: ekieff@ rics.bwh.harvard.edu.