Extensive Co-Operation between the Epstein-Barr Virus EBNA3 Proteins in the Manipulation of Host Gene Expression and Epigenetic Chromatin Modification

Abstract

Epstein-Barr virus (EBV) is able to drive the transformation of B-cells, resulting in the generation of lymphoblastoid cell lines (LCLs) in vitro. EBV nuclear proteins EBNA3A and EBNA3C are necessary for efficient transformation, while EBNA3B is dispensable. We describe a transcriptome analysis of BL31 cells infected with a series of EBNA3-knockout EBVs, including one deleted for all three EBNA3 genes. Using Affymetrix Exon 1.0 ST microarrays analysed with the MMBGX algorithm, we have identified over 1000 genes whose regulation by EBV requires one of the EBNA3s. Remarkably, a third of the genes identified require more than one EBNA3 for their regulation, predominantly EBNA3C co-operating with either EBNA3B, EBNA3A or both. The microarray was validated by real-time PCR, while ChIP analysis of a selection of co-operatively repressed promoters indicates a role for polycomb group complexes. Targets include genes involved in apoptosis, cell migration and B-cell differentiation, and show a highly significant but subtle alteration in genes involved in mitosis. In order to assess the relevance of the BL31 system to LCLs, we analysed the transcriptome of a set of EBNA3B knockout (3BKO) LCLs. Around a third of the genes whose expression level in LCLs was altered in the absence of EBNA3B were also altered in 3BKO-BL31 cell lines.Among these are TERT and TCL1A, implying that EBV-induced changes in the expression of these genes are not required for B-cell transformation. We also identify 26 genes that require both EBNA3A and EBNA3B for their regulation in LCLs. Together, this shows the complexity of the interaction between EBV and its host, whereby multiple EBNA3 proteins co-operate to modulate the behaviour of the host cell.

Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

“…3A). Previous reports have detected elevated ADAM 28 expression in EBV-negative BL cells (BL31) infected with a virus from which EBNA 3C has been deleted compared to wild-type virus-infected cells, implicating EBNA 3C as a repressor of ADAM28 (52). Our data further demonstrate that EBNA 3C can repress ADAM28 expression independently of other EBV latent proteins.…”

EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors

“…We next analyzed whether, in addition to more aggressive growth, B cells transformed with EBNA3BKO in vitro and in vivo share other phenotypic characteristics. We previously observed that the expression profile of EBNA3BKO-transformed B cells differs from that of their wtBAC-transformed counterparts, and many of the differentially expressed genes encode cell surface proteins (5). We confirmed the differential expression of 6 such genes by flow cytometry.…”

“…We generated LCLs with bacterial artificial chromosomederived (BAC-derived) EBNA3B-deleted EBV (referred to herein as EBNA3BKO) and identified genes that differed in expression from wild-type EBV-infected LCLs (ref. 5 and see below). These genes included a number of cell surface markers, chemokines, and potential oncogenes, which prompted us to investigate the impact of EBNA3B deletion in vivo.…”

“…We previously observed that more than 200 host genes exhibit altered expression levels in the absence of EBNA3B (5). To address the question of how these changes affect the in vivo biology of the virus, we used the recently established model of huNSG mice (NOD/SCID/γ c -/-mice transplanted with human hematopoietic progenitor cells [HPCs] to reconstitute components of the human immune system; refs.…”

“…Data analysis was performed using FlowJo analysis software (Tree Star Inc). Western blotting for EBV latency-associated antigens was performed using previously described antibodies (5).…”