Abstract
While the association of Epstein–Barr virus (EBV) with Burkitt lymphoma (BL) has long been recognised, the precise role of the virus in BL pathogenesis is not fully resolved. EBV can be lost spontaneously from some BL cell lines, and these EBV-loss lymphoma cells reportedly have a survival disadvantage. Here we have generated an extensive panel of EBV-loss clones from multiple BL backgrounds and examined their phenotype comparing them to their isogenic EBV-positive counterparts. We report that, while loss of EBV from BL cells is rare, it is consistently associated with an enhanced predisposition to undergo apoptosis and reduced tumorigenicity in vivo. Importantly, reinfection of EBV-loss clones with EBV, but surprisingly not transduction with individual BL-associated latent viral genes, restored protection from apoptosis. Expression profiling and functional analysis of apoptosis-related proteins and transcripts in BL cells revealed that EBV inhibits the upregulation of the proapoptotic BH3-only proteins, BIM and PUMA. We conclude that latent EBV genes cooperatively enhance the survival of BL cells by suppression of the intrinsic apoptosis pathway signalling via inhibition of the potent apoptosis initiators, BIM and PUMA.
Highlights
Epstein–Barr virus (EBV)-positive Burkitt lymphoma (BL), an aggressive and difficult to treat malignancy, is endemic in sub-Saharan Africa, where it accounts for around half of all childhood lymphomas
These clones were screened for EBV episome copy number by quantitative, real-time PCR (q-PCR); the results are summarised in Table 1 and Figure 2a
Before investigating the phenotype of the EBV-positive clones, their EBV infection status was fully characterised. This included flow cytometry for EBERs to confirm that all cells carried the virus, as well as immunoblotting and q-PCR for 45 EBV transcripts to ensure that the clones retained Latency I gene expression
Summary
Epstein–Barr virus (EBV)-positive Burkitt lymphoma (BL), an aggressive and difficult to treat malignancy, is endemic (eBL) in sub-Saharan Africa, where it accounts for around half of all childhood lymphomas. BIM, PUMA and tBID can directly activate BAX and BAK (reviewed in Strasser et al.[3]) It was shown using the Eμ-Myc transgenic mouse model of human BL (which expresses a c-Myc;Ig transgene) that blocking apoptosis through enforced expression of BCL-2 prosurvival proteins or deletion of BH3-only proteins or BAX greatly accelerates lymphoma development.[4,5,6,7]. A minority of eBLs exhibit a more complex viral gene expression pattern due to a genomic deletion in EBV.[9,10] Cell lines derived from these tumours show marked resistance to apoptosis due to epigenetic silencing of the BIM promoter[11] and functional inhibition of BIM, PUMA, BID and BAK by the viral BCL-2 homologue, BHRF1.12. EBV-positive and -negative BLs are genetically distinct, differing in terms of their cellular mutational profiles[13,14] and precise Ig-MYC chromosomal translocations.[15,16] It is unsatisfactory to introduce the virus or viral genes into EBV-negative spBL lines to study the role of EBV in eBL
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