Investigating the deregulation of host cell genes by Epstein-Barr virus nuclear antigens

Epstein-Barr virus (EBV) immortalises B-lymphocytes and is associated with the development of various human malignancies. The EBV transcription factors, Epstein-Barr nuclear antigens (EBNA)2, 3A, 3B and 3C drive B cell immortalisation through epigenetic reprogramming of cellular genes. Pathway analysis showed the B-cell receptor (BCR) signalling pathway to be enriched for EBNA-bound genes. We identified EBNA binding sites at promoter-proximal elements near BCR genes CD79A and CD79B and confirmed they are repressed by EBNA2. We found EBNA2 disrupts binding of transcription activator EBF1 at the CD79B promoter, correlating with decreased levels of acetylated histone H3, an active chromatin mark. NFATC1 and NFATC2, downstream effectors of the BCR signalling pathway, were also identified as EBNA target genes. We confirmed EBNA3B and 3C as negative regulators of NFATC1 and NFATC2, and EBNA2 as a negative regulator of NFATC1. However, we could not confirm direct binding by the EBNAs indicating repression may result from upstream deregulation. BCR stimulation results in release of the second messenger calcium. We found that Ca2+ release following BCR activation is significantly reduced in the presence of EBNA2. We also investigated how EBNA2 activates oncogene MYC and how EBNA3A and 3C silence pro-apoptotic gene BCL2L11; two key genes involved in growth regulation and lymphoma development. We found EBNA2 activation of MYC induced chromatin restructuring of the 3Mb MYC locus differently from ‘normal’ B cell activation via CD40; EBNA2 activation increased upstream, whereas CD40 activation increased downstream enhancer-promoter interactions. Additionally, EBNA2 recruits SWI/SNF to the MYC promoter where it is required to establish enhancer-promoter interactions. At BCL2L11 we showed EBNA3A and 3C disrupt enhancer-promoter interactions leading to PRC2 recruitment across the enhancer hub and deposition of the H3K27me3 silencing mark. Taken together these data shed light on the pathways and mechanisms through which EBV immortalises B cells and promotes lymphoma development.