Epstein-Barr Virus Nuclear Antigen 3c (Ebna3c) Interacts With The Metabolism Sensing C-Terminal Binding Protein (Ctbp) Repressor To Upregulate Host Genes

Author summaryEpstein-Barr virus (EBV) is a gammaherpesvirus that establishes lifelong infection in about 95% of adult humans. EBV infection is usually benign, but can rarely result in several different malignancies, particularly lymphomas. EBV infection of resting B-lymphocytes in the laboratory drives them to proliferate as lymphoblastoid cell lines (LCLs), a model for EBV lymphomagenesis. In this manuscript we study how one EBV protein expressed in LCLs, EBNA3C, contributes to B lymphocyte transformation. Prior work has established that EBNA3C turns off the CDKN2A gene, but there is disagreement regarding the relative importance of silencing the two CDKN2A gene products: p14 and p16. Using a CRISPR/Cas9 gene editing strategy we confirm that p16 knock-out rescues LCL growth in the absence of EBNA3C even in the presence of wildtype p14. We then use these knock-out LCLs to identify EBNA3C regulated genes and uncover extensive growth-independent changes in B lymphocytes due to the EBNA3C transcription factor. We also discover an unexpected role for the CtBP repressor protein in EBNA3C gene upregulation. Contrary to prior models, we do not observe CtBP recruitment to target genes by EBNA3C. Instead, our data are consistent with EBNA3C interfering with the ability of pre-bound CtBP to repress genes.Epstein-Barr virus (EBV) infection is associated with the development of specific types of lymphoma and some epithelial cancers. EBV infection of resting B-lymphocytes in vitro drives them to proliferate as lymphoblastoid cell lines (LCLs) and serves as a model for studying EBV lymphomagenesis. EBV nuclear antigen 3C (EBNA3C) is one of the genes required for LCL growth and previous work has suggested that suppression of the CDKN2A encoded tumor suppressor p16(INK4A) and possibly p14(ARF) is central to EBNA3C's role in this growth transformation. To directly assess whether loss of p16 and/or p14 was sufficient to explain EBNA3C growth effects, we used CRISPR/Cas9 to disrupt specific CDKN2A exons in EBV transformed LCLs. Disruption of p16 specific exon 1 alpha and the p16/p14 shared exon 2 were each sufficient to restore growth in the absence of EBNA3C. Using EBNA3C conditional LCLs knocked out for either exon 1 alpha or 2, we identified EBNA3C induced and repressed genes. By trans-complementing with EBNA3C mutants, we determined specific genes that require EBNA3C interaction with RBPJ or CtBP for their regulation. Unexpectedly, interaction with the CtBP repressor was required not only for repression, but also for EBNA3C induction of many host genes. Contrary to previously proposed models, we found that EBNA3C does not recruit CtBP to the promoters of these genes. Instead, our results suggest that CtBP is bound to these promoters in the absence of EBNA3C and that EBNA3C interaction with CtBP interferes with the repressive function of CtBP, leading to EBNA3C mediated upregulation.