Abstract
Epstein-Barr virus (EBV) oncoprotein EBNA3C is indispensable for primary B-cell transformation and maintenance of lymphoblastoid cells outgrowth. EBNA3C usurps two putative cellular pathways—cell-cycle and apoptosis, essentially through modulating ubiquitin-mediated protein-degradation or gene transcription. In cancer cells, these two pathways are interconnected with autophagy,—a survival-promoting catabolic network in which cytoplasmic material including mis/un-folded protein aggregates and damaged organelles along with intracellular pathogens are degraded and recycled in lysosomal compartments. Studies have shown that tumor viruses including EBV can manipulate autophagy as a survival strategy. Here, we demonstrate that EBNA3C elevates autophagy, which serves as a prerequisite for apoptotic inhibition and maintenance of cell growth. Using PCR based micro-array we show that EBNA3C globally accelerates autophagy gene transcription under growth limiting conditions. Reanalyzing the ENCODE ChIP-sequencing data (GSE52632 and GSE26386) followed by ChIP-PCR demonstrate that EBNA3C recruits several histone activation epigenetic marks (H3K4me1, H3K4me3, H3K9ac, and H3K27ac) for transcriptional activation of autophagy genes, notably ATG3, ATG5, and ATG7 responsible for autophagosome formation. Moreover, under growth limiting conditions EBNA3C further stimulates the autophagic response through upregulation of a number of tumor suppressor genes, notably cyclin-dependent kinase inhibitors—CDKN1B (p27Kip1) and CDKN2A (p16INK4a) and autophagy mediated cell-death modulators—DRAM1 and DAPK1. Together our data highlight a new role of an essential EBV oncoprotein in regulating autophagy cascade as a survival mechanism and offer novel-targets for potential therapeutic expansion against EBV induced B-cell lymphomas.
Highlights
Introduction EpsteinBarr virus (EBV) is a lymphotropic gammaherpesvirus that asymptomatically persists in >95% of the world population[1]
We asked whether EBNA3C mediated cell-cycle activation and apoptotic inhibition can be directly related to autophagy induction
lymphoblastoid cell-lines (LCLs) knockdown for EBNA3C38 and BJAB cells stably expressing EBNA3C (Fig. 1) were subjected to three different analyses—(i) western blots (WB) for cell-cycle, apoptosis (PARP cleavage) and autophagy (LC3II conversion and p62) markers; (ii) cell-proliferation assays, and (iii) cell-cycle analyses to determine subG0 fraction of resting/non-proliferating cells (Fig. 1 and S1)
Summary
Barr virus (EBV) is a lymphotropic gammaherpesvirus that asymptomatically persists in >95% of the world population[1]. In immuno-compromized individuals, the loss of immune-control of the latentlyinfected B-cells results in the emergence of several B-cell lymphomas[2,3,4]. EBV can efficiently transform resting B-lymphocytes into lymphoblastoid cell-lines (LCLs), providing an excellent model to study EBV-. Induced B-cell transformation and B-cell lymphomagenesis[5]. In LCLs, EBV expresses six nuclear antigens (EBNAs) and three membrane proteins (LMPs) along with several non-coding RNAs1,6. Five viral latent proteins including EBNA2, EBNA3A, EBNA3C, EBNALP, and LMP1 are indispensible for B-cell transformation and subsequent lymphoma development[6,7]. EBNA3C interferes with a number of cellular pathways —most strikingly cell-cycle/apoptosis and ubiquitin-
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