The Epstein-Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species

Abstract

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.

Fig. 1.

EBNA-1 induces genomic instability in B cells. (A) Metaphase chromosomes were analyzed in DG75 and BJAB (P), their sublines expressing stable EBNA-1 (E1), and BJAB transfectants expressing the tetracycline regulator alone (tTA) or tetracycline-regulated EBNA-1 (tTAE1) with and without treatment with 1 μg/ml tetracycline (Tet). EBNA-1 was detected in Western blots probed with the OT1X antibody, and β-actin was used as a loading control. (B) Comet assays were performed under neutral condition, and treatment with 1 μg of DNase was used as positive control. Representative micrographs illustrating the increased comet length in BJAB-E1 are shown. The mean ± SD comet length in 3 experiments is indicated in each panel. (C) Double-strand DNA breaks were quantified in BJAB-tTAE1 cells after EBNA-1 induction by removal of tetracycline (Left) or suppression by addition of tetracycline (Right). Minimal and maximal comet lengths were determined in BJAB-tTAE1 kept with or without tetracycline for at least 3 weeks. (D) pATM (red) and pH2AX (green) were visualized by immunofluorescence in cells untreated (Right) or pretreated for 18 h with 2 μg/ml etoposide (ETO) (Left). The nuclei were stained with DAPI (blue).

Fig. 2.

EBNA1 induces DNA damage and chromosomal aberrations via production of ROS. (A) The endogenous levels of ROS were determined by DCFDA staining in EBNA-1–positive and -negative BJAB (Top), in vitro EBV-converted BJAB and Ramos (Middle), and a panel of originally EBV-negative or -positive BL lines (Bottom). Representative FACS plots are shown for each cell line. The mean ± SD molecule equivalent of fluorescence (MEFL) of 3 experiments is shown. (B) Induction of DNA damage is abrogated by treatment with antioxidants. The cells were treated for 18 h with 3.5 μM ebselen (Ebs) or 1 mM citric acid (CA). Top:representative FACS plots and the mean ± SD MEFL of 3 independent experiments; Bottom: representative immunostaining for pH2AX. (C) Metaphase plates were analyzed in untreated cells and cells treated for the indicated time with 1.7 μM ebselen or 500 μM citric acid. Mean ± SD of 3 experiments.

Fig. 3.

EBNA-1 regulates the transcription of NOX2 and activation of the NADPH oxidase in EBV-positive cells. (A) The transcription levels of ACOX1, GPX1, NOX2, and GAPDH (control) were assayed by RT-PCR. NOX2 protein expression was visualized by Western blot. Beta-actin was used as loading control (Bottom). One representative experiment out of 3 performed with each cell line. (B) NOX2 was detected by Western blot in BJAB-tTAE1 after induction of EBNA-1 by removal of tetracycline. One representative experiment out of 3. (C) HL60 cells were cotransfected with the NOX2-Luc reporter and increasing amounts of the pCDNA3-FlagEBNA-1 plasmid. Relative luciferase activity was calculated as the ratio between the activity of NOX2-Luc and the maximal activity of a SV40-Luc reporter. All values were normalized to the activity of a cotransfected SV40-Renilla reporter. (D) EBNA-1 does not affect the expression of the NADPH oxidase subunits p22^phox, p47^phox, p67^phox, and Rac1. Western blots were probed with the indicated specific antibodies. One representative experiment out of 3. (E) Rac1GTP was detected by the G-Lisa Rac Activation Assay (Cytoskeleton). Where indicated, the cells were treated for 4 h with 1 μg/ml cytotoxic necrotizing factor (CNF). The mean ± SD of 3 experiments is shown. *, BJAB/BJAB-E1, P < 0.04; BJAB-tTAE1 ± tetracycline, P < 0.05.

Fig. 4.

Inhibition of the NADPH oxidase reverses the effect of EBNA-1. (A) Treatment with chemical inhibitors of the NADPH oxidase decreases the levels of ROS. The cells were pretreated with 100 μM Apo and/or 40 μM DPI for 18 h, and ROS activity was detected by DCFDA. Mean ± SD MEFL in 3 experiments. (B) Treatment with NADPH oxidase inhibitors decreases DNA damage. Neutral comet assays were performed in cells untreated or treated with Apo and DPI. Mean ± SD of comet length in 3 experiments. (C) Representative micrographs illustrating the decrease of pH2AX in cells treated with Apo and DPI. (D) BJAB-E1 cells were infected for 48 h with lentiviruses expressing nontargeting (nT) or NOX2-specific (cl.1, cl.2) shRNAs. NOX2 expression was detected by Western blots, and the intensity of the specific bands was quantified by densitometry. The mean ± SD of NOX2 levels in 3 experiments is shown. (E) NOX2 knockdown decreases the endogenous levels of ROS. ROS activity was detected by labeling with DCFDA. Mean ± SD fluorescence intensity in 3 experiments. (F) Representative micrographs illustrating the decreased pH2AX fluorescence after NOX2 knockdown are shown.