The Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 is the only viral protein expressed in all EBV-carrying malignancies, but its contribution to oncogenesis has remained enigmatic. We show that EBNA-1 induces chromosomal aberrations, DNA double-strand breaks, and engagement of the DNA damage response (DDR). These signs of genomic instability are associated with the production of reactive oxygen species (ROS) and are reversed by antioxidants. The catalytic subunit of the leukocyte NADPH oxidase, NOX2/gp91 phox , is transcriptionally activated in EBNA-1-expressing cells, whereas inactivation of the enzyme by chemical inhibitors or RNAi halts ROS production and DDR. These findings highlight a novel function of EBNA-1 and a possible mechanism by which expression of this viral protein could contribute to malignant transformation and tumor progression.is a human gamma-herpesvirus that establishes latent infections in B lymphocytes, where only a subset of viral genes is expressed and virus replication is suppressed (1). The proteins encoded by the latency genes, including 6 EBV-encoded nuclear antigens (EBNA-1, -2, -3A, -3B, -3C, and -5) and 3 latent membrane proteins (LMP1, -2A, and -2B), induce growth transformation by capturing multiple signaling pathways that control B cell proliferation and apoptosis. It is generally assumed that the continuous expression of viral genes underlies the association of EBV with a variety of human malignancies, including Burkitt's lymphoma (BL), Hodgkin's disease (HD), nasopharyngeal carcinoma (NPC), and posttransplant lymphoproliferative disease (PTLD) (2). Some EBVpositive tumors do not express all of the latency proteins, leading to restricted forms of latency in which EBNA-1 is detected either alone (latency I, found in BL) or together with the LMPs (latency II, found in HD and NPC). Thus, EBNA-1 is the only viral protein regularly expressed in all EBV-carrying malignancies.EBNA-1 binds to the viral origin of replication (oriP) and is required for the correct partitioning of the viral episomes in proliferating cells (3). It may confer a growth advantage to BL cells (4) and protect them from apoptosis (5) but does not act as an autonomous oncogene (6) and seems to be dispensable for B cell immortalization in vitro (7). Hence, the mechanism by which EBNA-1 may contribute to malignant transformation is not understood.Genomic instability is common in malignant cells and was observed in EBV-carrying tumors (8-10). EBNA-3C (11) and LMP-1 (12) may promote this phenotype through inhibition of DNA repair or inactivation of cell cycle checkpoints, which allow the propagation of DNA damage. However, these viral proteins are not expressed in EBV-carrying BLs, and only half of HDs and NPCs express detectable levels of LMP1, suggesting a limited role in EBV oncogenesis. A possible involvement of EBNA-1 in the induction of genomic instability is suggested by a significant increase of transient chromosomal aberrations, such as dicentric chromosomes, chromosome fragments, and gaps, in EBVpositiv...

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Epstein-Barr virus (EBV) has been implicated in the pathogenesis of human malignancies but the mechanisms of oncogenesis remain largely unknown. Genomic instability and chromosomal aberrations are hallmarks of malignant transformation. We report that EBV carriage promotes genomic instability in Burkitt's lymphoma (BL). Cytogenetic analysis of EBVÀ and EBV þ BL lines and their sublines derived by EBV conversion or spontaneous loss of the viral genome revealed a significant increase in dicentric chromosomes, chromosome fragments and chromatid gaps in EBV-carrying cells. Expression of EBV latency I was sufficient for this effect, whereas a stronger effect was observed in cells expressing latency III. Telomere analysis by fluorescent in situ hybridization revealed an overall increase of telomere size and prevalence of telomere fusion and double strand-break fusion in dicentric chromosomes from EBV þ cells. Phosphorylated H2AX, a reporter of DNA damage and ongoing repair, was increased in virus-carrying cells in the absence of exogenous stimuli, whereas efficient activation of DNA repair was observed in both EBV þ and EBVÀ cells following treatment with etoposide. These findings point to induction of telomere dysfunction and DNA damage as important mechanisms for EBV oncogenesis.

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