Epstein–Barr virus latent membrane protein-1 (LMP1) signalling is distinct from CD40 and involves physical cooperation of its two C-terminus functional regions

Abstract

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.

Multiple Carboxyl-Terminal Regions of the EBV Oncoprotein, Latent Membrane Protein 1, Cooperatively Regulate Signaling to B Lymphocytes Via TNF Receptor-Associated Factor (TRAF)-Dependent and TRAF-Independent Mechanisms

“…The stimulus is greater after supercrosslinking, suggesting that the physical proximity of the CTARs is important. Recent work supports the idea that CTAR-CTAR interactions are critical both by demonstrating that CTAR transcomplementation can occur in transiently transfected Jurkat T cells (24), and that the lytic LMP1 and a CTAR1-2 mutated LMP1 can function as inhibitors of LMP1 signaling in a dose-dependent manner (67,68). It is also relevant to note that simultaneous physical interactions between distinct regions of the CD40 cytoplasmic domain and TRAF3 have been proposed on the basis of studies of the crystal structure of a CD40 CT peptide complexed with a peptide of the TRAF3 C terminus (69).…”

“…The multitransmembrane domain LMP1 self-aggregates in the plasma membrane (15) and, as a result, has constitutive signaling activity (20). LMP1 mimics CD40 signaling in B cells (22,23), although important differences in LMP1 vs CD40 signaling in B cells and other cell types have been noted (24,25).…”

Roles of TNF Receptor-Associated Factor 3 in Signaling to B Lymphocytes by Carboxyl-Terminal Activating Regions 1 and 2 of the EBV-Encoded Oncoprotein Latent Membrane Protein 1

“…It has been previously shown in the Jurkat T cell line that CTAR1 and CTAR2 cooperate in signaling and that such cooperation requires physical association (either direct or indirect) within the same hetero-oligomeric complex (43). To test potential cooperation between CTARs in B cells, the principal target of EBV infection, our laboratory previously generated and examined B cell lines stably expressing hCD40CTAR1 and HLA-A2CTAR2 (composed of the extracellular and transmembrane domains of HLA-A2 and aa 242-386 of LMP1) (30).…”

Molecular Mechanisms of TNFR-associated Factor 6 (TRAF6) Utilization by the Oncogenic Viral Mimic of CD40, Latent Membrane Protein 1 (LMP1)

“…LMP1 and CD40 signaling result in kinase and NF-B activation and the up-regulation of costimulatory and adhesion molecules (12,18,27). However, LMP1 signals to B cells are amplified and sustained compared with CD40 signals (18,26).…”