ASSOCIATION OF TRAF1, TRAF2, AND TRAF3 WITH AN EPSTEIN-BARR-VIRUS LMP1 DOMAIN IMPORTANT FOR B-LYMPHOCYTE TRANSFORMATION - ROLE IN NF-KAPPA-B ACTIVATION

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-as sociated factors (TRAFs). We now show that in EBV-transformed B lympho cytes 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-lymph ocyte growth transformation (aa 187 to 231). Further deletional and al anine mutagenesis analyses and comparison with TRAF binding sequences in CD40, in CD30, and in the LMP1 of other lymphycryptoviruses provide the first evidence that PXQXT/S is a core TRAF binding motif. The neg ative effects of point mutations in the LMP1(1-231) core TRAF binding motif on TRAF binding and NF-kappa B activation genetically link the T RAFs to LMP1(1-231)-mediated NF-kappa B activation. NF-kappa B activat ion by LMP1(1-231) is likely to be mediated by TRAF1/TRAF2 heteroaggre gates since TRAF1 is unique among the TRAFs in coactivating NF-kappa B with LMP1(1-231), a TRAF2 dominant-negative mutant can block LMP1(1-2 31)-mediated NF-kappa B activation as well as TRAF1 coactivation, and 30% of TRAF2 is associated with TRAF1 in EBV-transformed B cells. TRAF 3 is a negative modulator of LMP1(1-231)-mediated NF-kappa B activatio n. Surprisingly, TRAF1, -2, or -3 does not interact with the terminal LMP1 CT aa 333 to 386 which can independently mediate NF-kappa B activ ation. The constitutive association of TRAFs with LMP1 through the aa 187 to 231 domain which is important in NF-kappa B activation and prim ary B-lymphocyte growth transformation implicates TRAF aggregation in LMP1 signaling.