Abstract
In human B cells infected with Epstein-Barr virus (EBV), latency-associated virus gene products inhibit expression of the pro-apoptotic Bcl-2-family member Bim and enhance cell survival. This involves the activities of the EBV nuclear proteins EBNA3A and EBNA3C and appears to be predominantly directed at regulating Bim mRNA synthesis, although post-transcriptional regulation of Bim has been reported. Here we show that protein and RNA stability make little or no contribution to the EBV-associated repression of Bim in latently infected B cells. However, treatment of cells with inhibitors of histone deacetylase (HDAC) and DNA methyltransferase (DNMT) enzymes indicated that epigenetic mechanisms are involved in the down-regulation of Bim. This was initially confirmed by chromatin immunoprecipitation analysis of histone acetylation levels on the Bim promoter. Consistent with this, methylation-specific PCR (MSP) and bisulphite sequencing of regions within the large CpG island located at the 5′ end of Bim revealed significant methylation of CpG dinucleotides in all EBV-positive, but not EBV-negative B cells examined. Genomic DNA samples exhibiting methylation of the Bim promoter included extracts from a series of explanted EBV-positive Burkitt's lymphoma (BL) biopsies. Subsequent analyses of the histone modification H3K27-Me3 (trimethylation of histone H3 lysine 27) and CpG methylation at loci throughout the Bim promoter suggest that in EBV-positive B cells repression of Bim is initially associated with this repressive epigenetic histone mark gradually followed by DNA methylation at CpG dinucleotides. We conclude that latent EBV initiates a chain of events that leads to epigenetic repression of the tumour suppressor gene Bim in infected B cells and their progeny. This reprogramming of B cells could have important implications for our understanding of EBV persistence and the pathogenesis of EBV-associated disease, in particular BL.
Highlights
Epstein-Barr virus (EBV) is a B lymphotropic gammaherpes virus that asymptomatically and persistently infects .90% of humans; it occasionally causes infectious mononucleosis in adolescents and in rare instances is associated with the development of several different types of B cell lymphoma and various epithelial tumours [1]
Bim is a cellular inducer of programmed cell death, so the level of Bim is a critical regulator of lymphocyte survival and reduced expression enhances lymphomagenesis in mice and humans
Regulation of Bim is uniquely important in the pathogenesis of Burkitt’s lymphoma (BL), since in this human childhood cancer the Myc gene is deregulated by chromosomal translocation and Myc can induce pcd via Bim
Summary
EBV is a B lymphotropic gammaherpes virus that asymptomatically and persistently infects .90% of humans; it occasionally causes infectious mononucleosis in adolescents and in rare instances is associated with the development of several different types of B cell lymphoma and various epithelial tumours [1]. In order to establish persistence, EBV infects non-dividing naıve B cells and drives these to proliferate as activated B blasts that express all the viral proteins found in LCLs. The transient expansion of an infected B blast population is accompanied by their differentiation, probably in germinal centres, to become centroblasts and centrocytes and resting memory B cells. The precise series of events that the EBV-positive B cells undergo to reach the memory compartment is not yet known It appears to involve the regulated shut-down and silencing of latent EBV gene expression from an initial state called latency III or the growth programme (as found in LCLs), via latency II ( known as the default programme), until in quiescent memory B cells no EBV
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