Role of the TRAF Binding Site and NF-kB Activation in Epstein-Barr Virus Latent Membrane Protein 1Induced Cell Gene Expression (original) (raw)
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Journal of Virology
In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-κB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/...
Journal of Biological Chemistry, 2003
Latent membrane protein 1 (LMP1), an Epstein-Barr virus transforming protein, is able to activate NF-B through its carboxyl-terminal activation region 1 (CTAR1) and 2 (CTAR2), but the exact role of each domain is not fully understood. Here we show that LMP1 activates NF-B in different NF-B essential modulator (NEMO)-defective cell lines, but not in cells lacking both IB kinase 1 (IKK1) and 2 (IKK2). Mutational studies reveal that CTAR1, but not CTAR2, mediates NEMOindependent NF-B activation and that this process largely depends on IKK1. Retroviral expression of LMP1 mutants in cells lacking either functional NF-B inducing kinase (NIK), NEMO, IKK1, or IKK2 further illustrates distinct signals from the two activation regions of LMP1 for persistent NF-B activation. One originates in CTAR2, operates through the canonical NEMO-dependent pathway, and induces NFKB2 p100 production; the second signal originates in CTAR1, utilizes NIK and IKK1, and induces the processing of p100. Our results thus help clarify how two functional domains of LMP1 persistently activate NF-B through distinct signaling pathways. Latent membrane protein-1 (LMP1) 1 is an oncogenic transmembrane protein encoded by Epstein-Barr virus (1-3) that is known to activate NF-B (4, 5), the c-Jun NH 2-terminal kinase pathway (6), and its downstream transcription factors such as AP-1 (6-8), Janus-activating tyrosine kinase 3 and signal transducer and activator of transcription (9), and p38 mitogenactivated protein kinase (10). Previous studies demonstrated that NF-B activation by LMP1 plays a pivotal role in its transforming activity (11-13). LMP1 is a constitutively active
Carolina Digital Repository (University of North Carolina at Chapel Hill), 1997
The Epstein-Barr virus (EBV)-encoded LMP1 protein induces several cellular changes including induction of epidermal growth factor receptor (EGFR) expression and activation of the NF-B transcription factor. Two domains within the carboxy terminus have been identified that activate NF-B. In this study, mutational analysis of the LMP1 protein indicated that the proximal NF-B activation domain, which is identical to the TRAF interaction domain (amino acids 187 to 231), is essential for induction of the EGFR. The distal NF-B activation domain (amino acids 352 to 386) did not induce expression of the EGFR. In contrast, the two domains both independently activated a B-CAT reporter gene and induced expression of the NF-B-regulated A20 gene in C33A epithelial cells. These results indicate that induction of the EGFR by LMP1 involves the TRAF interaction domain and that activation of NF-B alone is not sufficient. Northern blot analysis revealed that induction of EGFR and A20 expression is likely to be at the transcriptional level. Interestingly expression of CD40 in the C33A cells also induced expression of the EGFR. Overexpression of either TRAF3 or an amino-terminal-truncated form of TRAF3 (TRAF3-C) inhibited signaling from the LMP1 TRAF interaction domain but did not affect signaling from the distal NF-B activation domain. These data further define the mechanism by which LMP1 induces expression of the EGFR and indicate that TRAF signaling from LMP1 and CD40 activates a downstream transcription pathway distinct from NF-B that induces expression of the EGFR.
Epstein-Barr virus latent membrane protein 1 activation of NF- B through IRAK1 and TRAF6
Proceedings of the National Academy of Sciences, 2003
P rimary Epstein-Barr Virus (EBV) infection of B lymphocytes causes their long-term proliferation through expression of several proteins, including latent infection integral membrane protein 1 (LMP1). LMP1 is critical for EBV-infected cell activation, adhesion, and survival (1). EBV is causally associated with lymphoid and epithelial malignancies, including posttransplant lymphoproliferative disorders, Hodgkin's disease, anaplastic nasopharyngeal carcinoma, and gastric carcinomas; LMP1 is usually expressed in the malignant cells (2). LMP1 can transform rodent fibroblasts to anchorage, contact, and serum-independent growth and to tumorigenicity in nude mice (3). LMP1 expression in human B lymphoblasts alters cell growth, and transgenic expression in murine B cells causes hyperplasia and lymphoma (4).
Epstein–Barr virus latent membrane protein 1 activation of NF-κB through IRAK1 and TRAF6
Proceedings of the National Academy of Sciences, 2003
Epstein–Barr virus latent membrane protein 1 (LMP1) activation of NF-κB is critical for Epstein–Barr virus-infected B lymphocyte survival. LMP1 activates the IκB kinase complex and NF-κB through two cytoplasmic signaling domains that engage tumor necrosis factor receptor-associated factor (TRAF)1/2/3/5 or TRADD and RIP. We now use cells lacking expression of TRAF2, TRAF5, TRAF6, IKKα, IKKβ, IKKγ, TAB2, IL-1 receptor-associated kinase (IRAK)1, or IRAK4 to assess their roles in LMP1-mediated NF-κB activation. LMP1-induced RelA nuclear translocation was similar in IKKα knockout (KO) and WT murine embryo fibroblasts (MEFs) but substantially deficient in IKKβ KO MEFs. NF-κB-dependent promoter responses were also substantially deficient in IKKβ KO MEFs but were hyperactive in IKKα KO MEFs. More surprisingly, NF-κB responses were near normal in TRAF2 and TRAF5 double-KO MEFs, IKKγ KO MEFs, TAB2 KO MEFs, and IRAK4 KO MEFs but were highly deficient in TRAF6 KO MEFs and IRAK1 KO HEK293 cells....
Proceedings of the National Academy of Sciences, 2003
Epstein–Barr virus (EBV) latent infection membrane protein 1 (LMP1)-induced NF-κB activation is important for infected cell survival. LMP1 activates NF-κB, in part, by engaging tumor necrosis factor (TNF) receptor-associated factors (TRAFs), which also mediate NF-κB activation from LTβR and CD40. LTβR and CD40 activation of p100/NF-κB2 is now known to be NIK/IKKα-dependent and IKKβ/IKKγ independent. In the experiments described here, we found that EBV LMP1 induced p100/NF-κB2 processing in human lymphoblasts and HEK293 cells. LMP1-induced p100 processing was NIK/IKKα dependent and IKKβ/IKKγ independent. Furthermore, the LMP1 TRAF-binding site was required for p100 processing and p52 nuclear localization, whereas the LMP1 death domain-binding site was not. Moreover, the LMP1 TRAF-binding site preferentially caused RelB nuclear accumulation. In murine embryo fibroblasts (MEFs), IKKβ was essential for LMP1 up-regulation of macrophage inflammatory protein (MIP)-2, TNFα, I-TAC, ELC, MIG,...
Latent Membrane Protein 1 of Epstein-Barr Virus Induces CD83 by the NF-�B Signaling Pathway
J Virol, 2003
Epstein-Barr virus (EBV) infects human resting B cells and transforms them in vitro into continuously growing lymphoblastoid cell lines (LCLs). EBV nuclear antigen 2 (EBNA2) is one of the first viral proteins expressed after infection. It is able to transactivate viral as well as cellular target genes by interaction with cellular transcription factors. EBNA2 target genes can be studied easily by using an LCL (ER/EB2-5) in which wild-type EBNA2 is replaced by an estrogen-inducible EBNA2. Since the cell surface molecule CD83, a member of the immunoglobulin superfamily and a marker for mature dendritic cells, appeared on the surface of ER/EB2-5 cells within 3 h after the addition of estrogen, we analyzed the regulation of CD83 induction by EBV in more detail. Despite its rapid induction, CD83 turned out to be an indirect target gene of EBNA2. We could show that the viral latent membrane protein 1 (LMP1) is responsible for the induction of CD83 by using an LCL expressing a ligand-or antibody-inducible recombinant nerve growth factor receptor-LMP1 fusion protein. The inducibility of the CD83 promoter by LMP1 was mediated by the activation of NF-B, as seen by use of luciferase reporter assays using the CD83 promoter and LMP1 mutants. Additionally, fusion constructs of the transmembrane domain of LMP1 and the intracellular signaling domain of CD40, TNF-R1, and TNF-R2 likewise transactivated the CD83 promoter via NF-B. Our studies show that CD83 is also a target of the NF-B signaling pathway in B cells.