The Epstein-Barr virus Latent Membrane Protein-1 (LMP1) has structural features and functions consistent with it being a constitutively active cell surface receptor. The known association of LMP1 with members of the TRAF family of proteins suggests that LMP1 transduces signals similarly to the Tumour Necrosis Factor Receptor (TNFR) family of cell surface receptors that signal by forming dimers or trimers in response to binding of extracellular ligands. However, interactions between LMP1 and the TRAFs have so far only been described for the C-terminal activation region 1 (CTAR1) of LMP1 and no direct interactions of the TRAFs with the second NF-kB activation domain (CTAR2) have been reported. We have now mapped the NF-kB activation domain of CTAR2 to a highly conserved stretch of 6 amino acids at the far C-terminus (codons 379 to 384 in B95.8 LMP1). In addition, we constructed chimeric receptor molecules which contain the ligand-binding extracellular domain and the transmembrane domain of rat CD2 fused to the C-terminus of LMP1 encoding the CTAR1 and/or the CTAR2 domain. Interestingly, the function of a chimera encoding CTAR2 alone, as well as the function of a chimera encoding both CTAR1 and CTAR2 was found to be inducible upon antibody-mediated crosslinking. These inducible chimeric proteins also allowed us to demonstrate that LMP1 mediated NF-kB activation is an immediate event following activation of LMP1.

Tetracycline-regulated vectors were used to obtain inducible expression in stable transfected B cell lines of two Epstein-Barr virus (EBV) latent genes, LMP1 and EBNA2. The transfected genes were tightly repressed by low, nontoxic concentrations of tetracycline (< or = 1 microgram/ml) and, following removal of tetracycline, were induced to levels comparable to or up to 3x that of EBV-transformed normal lymphoblastoid cell lines. In transfected DG75 cells, induced expression of LMP1, but not of EBNA2, led to the expected upregulation of various cell surface markers, including: CD40, CD54, CD58, and HLA class I.A novel observation was that both LMP1 and EBNA2 independently caused the downregulation of surface IgM, an effect mirrored in EBV-positive Burkitt lymphoma lines undergoing phenotypic drift during the transition from latency I to latency III in which both LMP1 and EBNA2 are upregulated. Most remarkably, induced LMP1 expression almost completely inhibited cell growth for 4 to 5 days, after which the cells recovered a limited proliferative capacity. The cytostatic effect of LMP1 was observed in all three B cell lines studied: DG75, BJAB, and Akata. Further analysis showed that induction of LMP1 coincided with a reduction in the levels of c-myc, and that the cytostatic effect was due to an accumulation of cells at the G2/M phase of the cell cycle. These data suggest a novel function for the LMP1 oncogene in controlling the proliferation of EBV-infected cells by regulating progress through G2/M phase.

The Epstein ± Barr virus (EBV) encoded Latent Membrane Protein-1 (LMP1) mimics a constitutively active receptor molecule, and has been shown to activate NFkB and the MAPK and JNK pathways. Two regions within the cytosolic domain of LMP1 have been found to e ect cell signalling. One of these, the carboxy-terminal activation region-1 (CTAR1), binds members of the TRAF family of proteins, and the other (CTAR2) binds TRADD, suggesting that LMP1 transduces signals similarly to the Tumour Necrosis Factor Receptor family of receptors. The ability to bind TRAFs, to activate NFkB and the JNK pathway, to upregulate cellular genes such as CD54 (ICAM-1 adhesion molecule), and to a ect cell growth and apoptosis has led to the suggestion that LMP1 signalling is similar to, or even identical to CD40. However, we now show that while ligand-induced CD40 signalling is impaired in the Jurkat T cell line, LMP1 was fully functional; therefore demonstrating that LMP1 and CD40 signalling di er. Mutated LMP1 genes, in which one or other of the CTAR1 and CTAR2 domains was non-functional, behaved more like CD40 in being unable to upregulate the CD54 cell surface marker in Jurkat cells. However, the CTAR1 domain of LMP1, which shared a TRAF-binding sequence motif with CD40, di ered from CD40 in being unable to activate NF-kB in Jurkat. Cotransfection experiments with LMP1 mutants demonstrated that CTAR1 can cooperative with CTAR2 on separate LMP1 molecules, provided that they exist within the same oligomeric complex.

We wish to identify developmental changes in germinal center B cells that may contribute to their rapid growth. SHP-1 is an SH2 domain–containing phosphotyrosine phosphatase that negatively regulates activation of B cells and other cells of hematopoietic lineages. We have found that in all 13 EBV-negative and 11 EBV-positive Burkitt lymphomas with a nonlymphoblastoid phenotype, the mean concentration of SHP-1 was reduced to 5% of that of normal B and T cells. The possibility that this diminished expression of SHP-1 was related to the germinal center phenotype of Burkitt lymphomas was supported by the low to absent immunofluorescent staining for SHP-1 in germinal centers, and by the inverse relationship between the concentration of SHP-1 and the expression of the germinal center marker CD38 on purified tonsillar B cells. In CD38-high B cells, SHP-1 concentration was 20% of that of mantle zone B cells from the same donor. This reduction in SHP-1 is comparable to that of cells from motheaten viable mev/mev mice in which there is dysregulated, spontaneous signaling by cytokine and antigen receptors. Therefore, germinal center B cells may have a developmentally regulated, low threshold for cellular activation.