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

Abstract

Latent membrane protein 1 (LMP1) is an EBV-encoded transforming protein that strongly mimics the B cell-activating properties of a normal cellular membrane protein, CD40. LMP1 and CD40 both associate with the cytoplasmic adapter proteins called TNFR-associated factors (TRAFs). TRAFs 1, 2, and 3 bind to a region of LMP1 that is essential for EBV to transform B lymphocytes, carboxyl-terminal activating region (CTAR) 1. However, studies of transiently overexpressed LMP1 molecules, primarily in epithelial cells, indicated that a second region, CTAR2, is largely responsible for LMP1-mediated activation of NF-κB and c-Jun N-terminal kinase. To better understand LMP1 signaling in B lymphocytes, we performed a structure-function analysis of the LMP1 C-terminal cytoplasmic domain stably expressed in B cell lines. Our results demonstrate that LMP1-stimulated Ig production, surface molecule up-regulation, and NF-κB and c-Jun N-terminal kinase activation require both CTAR1 and CTAR2, and that these two regions may interact to mediate LMP1 signaling. Furthermore, we find that the function of CTAR1, but not CTAR2, correlates with TRAF binding and present evidence that as yet unidentified cytoplasmic proteins may associate with LMP1 to mediate some of its signaling activities.

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

“…To study LMP1 signaling, we have previously generated a chimeric molecule (hCD40LMP1) composed of the extracellular and transmembrane domains of hCD40 and the CCT of LMP1. This chimeric molecule signals indistinguishably from LMP1 but with controllable initiation, and like WT LMP1, its aggregation localizes the hybrid receptor to plasma membrane rafts (24,29,30). The extracellular domains of hCD40 and mCD40 are not cross-reactive with species-specific Abs, so hCD40LMP1 and the endogenous mCD40 can be engaged differentially (24,29,30).…”

“…This chimeric molecule signals indistinguishably from LMP1 but with controllable initiation, and like WT LMP1, its aggregation localizes the hybrid receptor to plasma membrane rafts (24,29,30). The extracellular domains of hCD40 and mCD40 are not cross-reactive with species-specific Abs, so hCD40LMP1 and the endogenous mCD40 can be engaged differentially (24,29,30). To dissect the signaling mediated by the two functional domains of the CCT of LMP1, our laboratory has also constructed two deletion mutants, hCD40CTAR1 (aa 187-241 of LMP1) and hCD40CTAR2 (aa 242-386 of LMP1), within the context of hCD40LMP1 chimeric molecule (Fig.…”

“…We thus wished to explore further how TRAF3 regulates LMP1-mediated signaling to B cells. Our previous studies revealed that both CTAR1 and CTAR2 are equally important in mediating LMP1-induced signaling in B cells (30). The present study examines how TRAF3 interacts with these LMP1 signaling regions to regulate B cell activation.…”

“…DNA constructs used to stably express hCD40LMP1 and its mutants, including hCD40CTAR1, hCD40CTAR2, hCD40PQAA1, hCD40PQAA2, and hCD40PQAA3 have been described previously (24,30). The hCD40LMP1 mutant hCD40YYD3A was generated by PCR using hCD40 -5Ј (5Ј-AAG TCG ACG CCT CGC TCG GGC GCC A-3Ј) and LMP1YYD3A (5Ј-CTA GTC TAG AAG GTT AGG CTG CTG CGC TTA GCT GAA CTG GGC CGT-3Ј) as primers and hCD40LMP1 as template.…”

“…For coexpression of hCD40CTAR1 and hCD40CTAR2, CH12.LX cells were first transfected with hCD40CTAR1.pRSV.5(Neo) and selected with 400 g/ml geneticin. Surface expression of hCD40CTAR1 was determined by immunofluorescence flow cytometry as previously described (24,30). Clones stably expressing hCD40CTAR1 were then supertransfected with hCD40CTAR2.pRSV.5(Zeo) and selected with BCM-10 containing 400 g/ml Zeocin and 400 g/ml geneticin.…”