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. 2011 Jul;85(13):6764-73.
doi: 10.1128/JVI.00422-11. Epub 2011 May 4.

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

Benjamin E Gewurz  1 , Jessica C Mar, Megha Padi, Bo Zhao, Nicholas P Shinners, Kaoru Takasaki, Edward Bedoya, James Y Zou, Ellen Cahir-McFarland, John Quackenbush, Elliott Kieff
Affiliations

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

Benjamin E Gewurz et al. J Virol. 2011 Jul.

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-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. 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-α) 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 IκBα and A20. LMP1 TES2-regulated RNAs encode many NF-κB 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 IκBα superrepressor coexpression decreased LMP1 TES2 RNA effects to only 5 RNAs, with FDRs of <0.001-fold and >2-fold changes. Thus, canonical NF-κB activation is critical for almost all LMP1 TES2 RNA effects in HEK-293 cells and a more significant therapeutic target than previously appreciated.

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Figures

Fig. 1.
Fig. 1.
LMP1 TES2-inducible expression activates p38, ERK, JNK, and NF-κB pathways with similar kinetics. Western blot assays were performed with phospho-specific antibodies to p38 (serines 180 and 182), ERK (serines 202 and 204), JNK (serines 183 and 185), and RelA (serine 536). LMP1 TES2 expression was induced at time zero. Lysates from TNF-α-treated cells (10 ng/ml for 15 min) are shown as a positive control.
Fig. 2.
Fig. 2.
LMP1 TES2 significantly alters the abundance of numerous cell RNAs. (A) Volcano plot showing effects of 24 h of LMP1 TES2 expression on cell RNAs. Each cell RNA is represented by a dot in the 2-dimensional space. The x axis is defined by the log2 fold change in gene expression at 0 versus 24 h, and the y axis by is the log-adjusted P value. (B) Western blot analysis of LMP1 TES2 expression across the time course.
Fig. 3.
Fig. 3.
LMP1 TES2 strongly upregulates RNAs that encode the NF-κB inhibitors IκBα and A20. RNA was prepared in triplicate from cells induced for LMP1 TES2 expression for 0, 6, 9, 12, and 24 h. Log2 RMA-normalized RNA levels are shown on the y axis.
Fig. 4.
Fig. 4.
LMP1 TES2 regulates RNAs that encode multiple NF-κB pathway components and their direct protein-protein interactors. Core NF-κB pathway components (based on Biocarta) are depicted as diamonds. Edges indicate protein-protein interactions (Biogrid) between NF-κB pathway components and LMP1 TES2 targets. Nodes are shaded by the magnitude of TES2 effects; upregulated and downregulated targets are shaded red and blue, respectively. HUGO nomenclature was used.
Fig. 5.
Fig. 5.
Agglomerative hierarchical clustering analysis revealed distinct temporal patterns of LMP1 TES2 target gene regulation. Triplicate data are shown for each time point.
Fig. 6.
Fig. 6.
LMP1 TES2 activates p38, JNK, and ERK in the presence of an IκBα superrepressor (IκBαSR). Data for Western blot analysis of LMP1 TES2-induced MAPK phosphorylation, degradation of endogenous IκBα, and IκBαSR expression across the time course are shown. LMP1 TES2 and IκBαSR expression were induced at time zero.
Fig. 7.
Fig. 7.
LMP1 TES2 requires canonical NF-κB activity to regulate essentially all cell target genes. (A) Volcano plot showing effects of 24 h of LMP1 TES2 expression on cell gene transcription in the presence of an IκBα superrepressor (SR). (B) Western blot analysis of LMP1 TES2 expression in profiled samples.
Fig. 8.
Fig. 8.
LMP1 TES2 requires NF-κB activity to upregulate target genes. Median-centered time course expression profile data are shown for the top 20 LMP1 TES2-upregulated RNAs in the absence (left) or presence (right) of an IκBα superrepressor (SR). *, the IκBαSR message is recognized by the IκBα microarray probe set (gene symbol, NFKBIA). HUGO nomenclature was used.
Fig. 9.
Fig. 9.
LMP1 TES2 requires NF-κB activity to downregulate target genes. Median-centered time course expression profile data are shown for the top 20 LMP1 TES2-downregulated RNAs in the absence (left) or presence (right) of an IκBα superrepressor (SR). HUGO nomenclature was used.
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