Epstein–Barr Virus B Cell Growth Transformation: The Nuclear Events

Epstein–Barr virus (EBV) is the first human DNA tumor virus identified from African Burkitt’s lymphoma cells. EBV causes ~200,000 various cancers world-wide each year. EBV-associated cancers express latent EBV proteins, EBV nuclear antigens (EBNAs), and latent membrane proteins (LMPs). EBNA1 tethers EBV episomes to the chromosome during mitosis to ensure episomes are divided evenly between daughter cells. EBNA2 is the major EBV latency transcription activator. It activates the expression of other EBNAs and LMPs. It also activates MYC through enhancers 400–500 kb upstream to provide proliferation signals. EBNALP co-activates with EBNA2. EBNA3A/C represses CDKN2A to prevent senescence. LMP1 activates NF-κB to prevent apoptosis. The coordinated activity of EBV proteins in the nucleus allows efficient transformation of primary resting B lymphocytes into immortalized lymphoblastoid cell lines in vitro.


Introduction
Epstein-Barr virus (EBV) was discovered from African Burkitt's lymphoma (BL) cells over fifty years ago [1]. EBV causes many different cancers including B cell lymphomas and Hodgkin's lymphoma in people with T-cell immune suppression due to HIV infection, transplantation, or advanced age; nasopharyngeal carcinoma; and~10% of gastric cancers [2]. During primary EBV infection, viruses replicate in oro-pharyngeal epithelial cells and infect resting B-lymphocytes (RBLs). Latency III EBV nuclear antigens, EBNA2, leader protein (EBNALP), EBNA3A, EBNA3C, and Latent Membrane Protein 1 are the principal causes of infected B cell proliferation. While immune responses eliminate most B cells expressing latency III proteins, escaped cells enter lymphatic tissues, where EBV protein expression is down-regulated and only EBNA1 is expressed. EBV infected cells that escape immune destruction are sites of long-term latency persistence, reactivation, and can evolve to EBV+ B cell lymphomas and Hodgkin's lymphoma [3].
EBV immortalizes RBLs to lymphoblastoid cell lines (LCLs) in vitro. LCLs express type III EBV latency genes. Post-transplant lymphoproliferative disease (PTLD) and AIDS CNS lymphomas express the same latency III EBV genes as LCLs. Therefore, LCLs are a useful and appropriate model for genetic and biochemical investigation of EBV's roles in converting RBLs to proliferating lymphoblasts in vivo.

EBV Transformation from RBL to LCL Timeline
EBV infection of RBLs in vitro results in the establishment of LCLs in 3-4 weeks. Once established, LCLs can be kept in culture and grow continuously. Two days after EBV infection of RBLs, the cell size starts to increase and continues to increase. By day 4, cell size reaches the peak and starts to decrease slowly. Eight days after infection, the cell size is approximately two times the size of RBLs [4,5]. EBNA mRNAs are expressed at LCL level by day 2 and maintain a similar level afterwards. LMP1 mRNA level starts to increase two days after infection and gradually increases to LCL level at day 28 [6][7][8]. EBNA2 protein RNA POLII ChIA-PET links all the EBNA2 enhancers to their direct target genes genomewide [54]. Most importantly, ChIA-PET links EBNA2 enhancers to MYC TSS, in agreement with data generated by capture genome-wide 3C followed by deep sequencing (C Hi-C) and circular 3C followed by deep sequencing (4C-seq) [55]. The EBNA2 enhancer interaction patterns are extremely complicated. These enhancers are found both upstream and downstream of their direct target genes. Some enhancers interact with only one target gene. Some genes interact with multiple enhancers. Some enhancers interact with multiple genes. Some enhancers skip the nearest gene to interact with genes further away. CRISPR disruption of RBPJ sites within the EBNA2 enhancer greatly reduces the mRNA levels of EBNA2-enhancer-linked genes [56].
Host enhancer/silencer rewiring following EBV transformation of RBLs into LCLs. In RBLs, MYC and CCND2 are expressed at basal level with few enhancer promoter interactions. p16INK4A is expressed at high level with extensive enhancer-promoter interactions and active enhancer mark H3K27ac. Following EBV transformation, EBNA2 induces enhancers 400-500 kb upstream of MYC to loop to MYC TSS to activate MYC expression. EBNALP recruits YY1 to CCND2 locus and promotes enhancer-promoter interaction to activate CCND2 expression. These loci are now marked by H3K27ac. At the p16INK4A locus, much less enhancer-promoter interactions is seen and EBNA3C recruits transcription repressors Sin3A and polycomb repressive complexes to the locus with increased H3K27me3. EBNA3C stabilizes CCND and increases CDK6 kinase activity.
Super-enhancers (SEs) are clusters of enhancers with extraordinary broad and high active histone marks, including H3K27ac and H3K4me1 or TF ChIP-seq peaks [57]. SEs play critical roles in development, differentiation, and oncogenesis [57]. EBNA2 SEs are enhancers with extraordinary broad and tall EBNA2 ChIP-seq peaks [54]. EBNA2 SEs control the expression of cell genes that are critically important for cell proliferation, including MYC, MAX, RUNX3, BCL2, EBF, BATF, etc. [54,58]. CRISPR deletion of MYC SE using paired sgRNAs targeting the edges of EBNA2 -525 SE greatly reduces MYC expression and cell growth in LCLs [56]. In addition to EBNA2 binding, EBNA2 SEs are also bound by many B cell TFs, including BRD4, EP300, RNA POLII, BATF, EBF, IRF4, etc. [54]. The Figure 1. Host enhancer/silencer rewiring following EBV transformation of RBLs into LCLs. In RBLs, MYC and CCND2 are expressed at basal level with few enhancer promoter interactions. p16INK4A is expressed at high level with extensive enhancer-promoter interactions and active enhancer mark H3K27ac. Following EBV transformation, EBNA2 induces enhancers 400-500 kb upstream of MYC to loop to MYC TSS to activate MYC expression. EBNALP recruits YY1 to CCND2 locus and promotes enhancer-promoter interaction to activate CCND2 expression. These loci are now marked by H3K27ac. At the p16INK4A locus, much less enhancer-promoter interactions is seen and EBNA3C recruits transcription repressors Sin3A and polycomb repressive complexes to the locus with increased H3K27me3. EBNA3C stabilizes CCND and increases CDK6 kinase activity.
Super-enhancers (SEs) are clusters of enhancers with extraordinary broad and high active histone marks, including H3K27ac and H3K4me1 or TF ChIP-seq peaks [57]. SEs play critical roles in development, differentiation, and oncogenesis [57]. EBNA2 SEs are enhancers with extraordinary broad and tall EBNA2 ChIP-seq peaks [54]. EBNA2 SEs control the expression of cell genes that are critically important for cell proliferation, including MYC, MAX, RUNX3, BCL2, EBF, BATF, etc. [54,58]. CRISPR deletion of MYC SE using paired sgRNAs targeting the edges of EBNA2 -525 SE greatly reduces MYC expression and cell growth in LCLs [56]. In addition to EBNA2 binding, EBNA2 SEs are also bound by many B cell TFs, including BRD4, EP300, RNA POLII, BATF, EBF, IRF4, etc. [54]. The enrichment of activated and basal TFs in SEs facilitates the phase separation that allows the formation of condensates that robustly increase the transcription activity [59,60].

EBNALP in Transcription Regulation
The AA sequence of EBNALP consists of various numbers of identical W repeats and unique Y exons in different EBV isolates. Two copies of W repeats are the minimum for EBNALP to function [61]. Four copies of W repeats are enough for EBNALP to exert optimum activity [62]. A genetic study inserting a stop codon to the end of the last W repeat indicated that EBNALP is important for EBV transformation of RBLs [63]. Mutant EBV with stop codons inserted into every W exon fails to transform naïve B cells from the cord blood [64], indicating that EBNALP is essential for EBV transformation of naïve B cells. This mutant EBV transforms memory B cells with reduced efficiency [4,64].
EBNALP does not bind to DNA directly, instead it is tethered to DNA through host proteins. EBNALP ChIP-seq finds that EBNALP binds to thousands of promoter and enhancer sites [75]. Interestingly,~33% of EBNALP binding sites are at promoters. In contrast, only~14% of the EBNA2 binding sites are at promoters [19]. EBNALP peaks are also different from EBNA2 peaks that are very sharp and tall. EBNALP peaks tend to be much wider and lower. Integrating the ENCODE GM12878 ChIP-seq data with EBNALP data finds that 82% of the EBNALP peaks overlap with DPF2 ChIP-seq peaks. DPF2 binds to H3K14ac and H4K16ac and can bridge EBNALP to chromatin [76]. Motif analysis identifies that the motifs enrich at the EBNALP ChIP-seq peaks, including CTCF, ETS, IRF4, YY1. A total of 59% of EBNALP peaks overlaps with YY1 peaks in LCLs [75]. YY1 also binds preferentially to promoters, and it bridges promoter-enhancer interactions [77]. The high degree of overlap between EBNALP and YY1 peaks in LCLs suggests that EBNALP may play important role in mediating host genome organization (Figure 1).

EBNA3A and EBNA3C
EBNA3A, 3B, and 3C are located in tandem in the EBV genome. They all have similar gene structure with a short exon and a long exon. It is believed that they are the products of gene duplication. They share limited homology in their N-terminal AA sequences that mediate their interactions with host TF RBPJ that tethers EBNA2 to DNA [78][79][80]. Genetic studies indicate that EBNA3B is dispensable while EBNA3A is important and EBNA3C is essential for LCL growth [4,[81][82][83][84]. EBNA3A AAs 170-240 and 300-523 are important for LCL growth [85]. EBNA3C AAs 50-400 and 800-900 are essential for LCL growth [86,87].
Repression of p16 INK4A is the most important function of EBNA3C. shRNA knockdown of p16 INK4A and p14 ARF allows LCLs expressing conditional EBNA3C to grow in the non-permissive condition. EBNA3C inactivation does not affect the DNA methylation profiles at the loci but increases H3K27me3 and decreases H3K4me1 [84,100]. EBNA3C null virus can transform B cells from primary B cells from an individual with homozygous deletion of p16 INK4a [110]. CRISPR knockout of p16 INK4A also allows LCLs to grow in the absence of EBNA3C [111]. EBNA3C binds to p14 ARF promoter and recruits Sin3A repressor to the site. The Sin3A recruitment is dependent on EBNA3C expression [112]. EBNA3C interactions with RBPJ, CtBP, WDR48, and USP46 are also important for EBNA3C-mediated p16 INK4A repression [92,93,111] (Figure 1). EBNA3C maintains the LMP1 expression level in growth-arrested Raji cells [113]. EBNA3C co-activates the LMP1 promoter together with EBNA2 [114,115]. EBNA3C upregulates the expression of AID that is important for somatic hypermutation and class switch [116,117].

LMP1
LMP1 activates canonical and non-canonical NF-κB pathways [148]. NF-κB family TFs have five subunits that can form a heterodimer or homodimers and bind to DNA [149]. These proteins are each critical for B cell development and function. NF-κB subunit ChIP-seq identifies a complex NF-κB-binding landscape in LCLs. Nearly one-third of NF-κB-binding sites lack κB motifs and are instead enriched for alternative motifs. The oncogenic forkhead box protein FOXM1 co-occupies nearly half of NF-κB-binding sites and forms protein complexes with NF-κB on DNA. FOXM1 knockdown decreases NF-κB target gene expression and ultimately induces apoptosis, highlighting FOXM1 as a synthetic lethal target in B cell malignancy [149].

Future Directions
The expression of viral transcription factors in EBV cancer provides a unique opportunity for targeted therapies. Traditionally, TFs are not ideal drug targets. However, the development of proteolysis targeting chimeras (PROTACs) now allows specific degradation of TFs [150]. A small molecule that binds to TFs is linked to E3 ligase ligand. The molecule then recruits E3 ligase to the TF and degrades the TF. PROTACs have been developed to target many TFs and the degradation of TFs important for cancer cells can inhibit cell growth [151][152][153]. It is therefore now possible to develop EBV-specific PROTACs to degrade EBV TFs to treat EBV-associated cancers.