Dataset Information

Canonical NF-kappaB activation is essential for Epstein-Barr virus latent membrane protein 1 TES2/CTAR2 gene regulation.

ABSTRACT: Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) transforms rodent fibroblasts and is expressed in most EBV-associated malignancies. LMP1 (transformation effector site 2 [TES2]/C-terminal activation region 2 [CTAR2]) activates NF-?B, p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and interferon regulatory factor 7 (IRF7) pathways. We have investigated LMP1 TES2 genome-wide RNA effects at 4 time points after LMP1 TES2 expression in HEK-293 cells. By using a false discovery rate (FDR) of <0.001 after correction for multiple hypotheses, LMP1 TES2 caused >2-fold changes in 1,916 mRNAs; 1,479 RNAs were upregulated and 437 were downregulated. In contrast to tumor necrosis factor alpha (TNF-?) stimulation, which transiently upregulates many target genes, LMP1 TES2 maintained most RNA effects through the time course, despite robust and sustained induction of negative feedback regulators, such as I?B? and A20. LMP1 TES2-regulated RNAs encode many NF-?B signaling proteins and secondary interacting proteins. Consequently, many LMP1 TES2-regulated RNAs encode proteins that form an extensive interactome. Gene set enrichment analyses found LMP1 TES2-upregulated genes to be significantly enriched for pathways in cancer, B- and T-cell receptor signaling, and Toll-like receptor signaling. Surprisingly, LMP1 TES2 and I?B? superrepressor coexpression decreased LMP1 TES2 RNA effects to only 5 RNAs, with FDRs of <0.001-fold and >2-fold changes. Thus, canonical NF-?B activation is critical for almost all LMP1 TES2 RNA effects in HEK-293 cells and a more significant therapeutic target than previously appreciated.

SUBMITTER: Gewurz BE

PROVIDER: S-EPMC3126495 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Canonical NF-kappaB activation is essential for Epstein-Barr virus latent membrane protein 1 TES2/CTAR2 gene regulation.

Gewurz Benjamin E BE Mar Jessica C JC Padi Megha M Zhao Bo B Shinners Nicholas P NP Takasaki Kaoru K Bedoya Edward E Zou James Y JY Cahir-McFarland Ellen E Quackenbush John J Kieff Elliott E

Journal of virology 20110504 13

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) transforms rodent fibroblasts and is expressed in most EBV-associated malignancies. LMP1 (transformation effector site 2 [TES2]/C-terminal activation region 2 [CTAR2]) activates NF-κB, p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and interferon regulatory factor 7 (IRF7) pathways. We have investigated LMP1 TES2 genome-wide RNA effects at 4 time points after LMP1 TES2 expression in HEK-293 cells. B ...[more]

PMID: 21543491

Similar Datasets

Project description:Epstein-Barr Virus (EBV) Latent Membrane Protein 1 (LMP1) transforms rodent fibroblasts and is expressed in most EBV-associated malignancies. LMP1 Transformation Effector Site 2 (TES2)/C-Terminal Activation Region 2 (CTAR2) activates NF-kappaB, p38, JNK, ERK and IRF7 pathways. We have investigated LMP1 TES2 genome-wide RNA effects at 4 time points after LMP1 TES2 expression in HEK 293 cells. Using a False Discovery Rate (FDR) of < 0.001 after correction for multiple hypotheses, LMP1 TES2 caused > 2-fold changes in 1916 mRNAs; 1479 RNAs were up-regulated and 437 down-regulated. In contrast to TNFalpha stimulation, which transiently up-regulates many target genes, LMP1 TES2 maintained most RNA effects through the time course, despite robust and sustained induction of negative feedback regulators, such as IkappaBalpha and A20. LMP1 TES2 regulated RNAs encode many NF-kappaB signaling proteins and secondary interacting proteins. Consequently, many LMP1 TES2-regulated RNAs encode proteins that form an extensive interactome. Gene Set Enrichment Analyses found LMP1 TES2 up-regulated genes to be significantly enriched for Pathways in Cancer, B-and T-cell receptor signaling, and Toll-like receptor signaling. Surprisingly, LMP1 TES2 and IkappaBalpha super-repressor co-expression decreased LMP1 TES2 RNA effects to only 5 RNAs with FDR<0.001 and >2 fold change. Thus, canonical NF-kappaB activation is critical for almost all LMP1 TES2 RNA effects in HEK-293 cells and a more significant therapeutic target than previously appreciated. An LMP1 double point mutant (P204A, Q206A) was used to construct a HEK-293 TET-On LMP1 TES2 cell line. Stable cell clones were selected that carry an inducible system for LMP1 TES2 expression. The Tet-system is composed of three parts: 1) the LMP1 TES2 cDNA cloned into the tetracycline-regulated pJEF vector; 2) a tetracycline suppressor (tTS) that binds Tet-operator sites in the absence of tetracyclines and silences expression; 3) a reverse-tetracycline transactivator fused to the 4-hydroxy tamoxifen (4HT) ligand binding domain (rTTA M2). LMP1 expression was induced by addition of doxycycline (1 ug/ml) and 4HT (100 nM). For simultaneous inducible expression of LMP1 TES2 and an IkBa super-repressor, a stable cell line was derived. A pJEF vector encoding IkBa residues 37-317 was introduced into the inducible LMP1 TES2 cell line. Cell lines were cultured with DMEM (GIBCO) supplemented with 10% tetracycline-free serum (Clontech). LMP1 expression was induced by addition of tetracycline (1 ug/ml). RNA samples were collected from triplicate samples using RNABee (Qiagen) according to the manufacturer’s instructions from 293 cells induced for LMP1 TES2 (and IkBa super-repressor, where indicated) expression at 0, 6, 9, 12 and 24 hours of induction. Cells were in the log-phase of growth. Gene expression profiles were assayed using the Affymetrix HU-133 plus2 GeneChip according to the manufacturer's instructions. Real-time RT-PCR was performed with the Power SYBR Green RNA-to-CT™ 1-Step Kit (Applied Biosystems, Foster City, CA). Fold changes were determined using delta delta Ct method and normalized by GAPDH expression levels. RNA expression data were normalized using RMA and array quality assessed by NUSE and RLE scores. The data was collected according to methods that fall under the MIAME standards.

Project description:BackgroundThe Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB.ResultsIn this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied.ConclusionsInhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.

Project description:Transporter associated with antigen processing 2 (Tap-2) is responsible for ATP-dependent transport of peptides from the cytosol to the endoplasmic reticulum, where peptides bind to newly synthesized human leukocyte antigen (HLA) class I molecules, which are essential for cellular immune responses. Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) has been shown to induce the expression of Tap-2. In this study, the induction of endogenous Tap-2 by LMP-1 is shown to be associated with and requires the expression of interferon regulatory factor 7 (IRF-7). In DG75 Burkitt's lymphoma (BL) cells, in which LMP-1 induces the expression of IRF-7, LMP-1 induced endogenous Tap-2, and ectopic expression of IRF-7 could enhance the induction. In Akata BL cells, in which LMP-1 could not induce IRF-7, LMP-1 could not induce Tap-2. Addition of IRF-7, which complements the defect in Akata cells, could stimulate the expression of Tap-2. Furthermore, LMP-1 and IRF-7A but not other IRF-7 splicing variants could activate endogenous Tap-2. A Tap-2 promoter reporter construct could be activated by the overexpression of IRF-7A. The activation could be specifically enhanced by LMP-1 and was dependent on an intact interferon-stimulated response element (ISRE) present in the Tap-2 promoter. Also, IRF-7 can bind to the Tap-2 promoter under physiological conditions in vivo, as shown by formaldehyde cross-linking, as well as to the Tap-2 ISRE in vitro, as shown by gel mobility shift assays. Furthermore, LMP-1 facilitates the phosphorylation and nuclear translocation of IRF-7. These data point to the role of IRF-7 as a secondary mediator of LMP-1-activated signal transduction for Tap-2 as follows: LMP-1 stimulates the expression of IRF-7 and facilitates its phosphorylation and nuclear translocation, and then the activated IRF-7 mediates the activation of the cellular Tap-2 gene. The induction of Tap-2 by IRF-7 and LMP-1 may have an important implication for the immune response to EBV and its persistence in vivo.

Dataset Information

Canonical NF-kappaB activation is essential for Epstein-Barr virus latent membrane protein 1 TES2/CTAR2 gene regulation.

Publications

Canonical NF-kappaB activation is essential for Epstein-Barr virus latent membrane protein 1 TES2/CTAR2 gene regulation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets