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. 2004 Nov;78(22):12694-7.
doi: 10.1128/JVI.78.22.12694-12697.2004.

EBNA2 is required for protection of latently Epstein-Barr virus-infected B cells against specific apoptotic stimuli

Jae Myun Lee  1 , Kyoung-Ho Lee, Christopher J Farrell, Paul D Ling, Bettina Kempkes, Jeon Han Park, S Diane Hayward
Affiliations

EBNA2 is required for protection of latently Epstein-Barr virus-infected B cells against specific apoptotic stimuli

Jae Myun Lee et al. J Virol. 2004 Nov.

Abstract

In addition to functioning as a transcriptional transactivator, Epstein-Barr virus EBNA2 interacts with Nur77 to protect against Nur77-mediated apoptosis. Estrogen-regulated EBNA2 in EREB2-5 cells was replaced by either EBNA2 or EBNA2 with a deletion of conserved region 4 (EBNA2DeltaCR4). Both EBNA2-converted and EBNA2DeltaCR4-converted EREB2-5 cells grew in the absence of estrogen and expressed LMP1. Treatment with tumor necrosis factor alpha did not induce apoptosis of EBNA2- or EBNA2DeltaCR4-expressing cells, but EBNA2DeltaCR4 cells were susceptible to etoposide and 5-fluorouracil, Nur77-mediated inducers of apoptosis. Thus, EBNA2 protects B cells against specific apoptotic agents against which LMP1 is not effective.

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Figures

FIG. 1.
FIG. 1.
Effect of estrogen starvation on growth of EBNA2-transduced EREB2-5 cells. Ten thousand cells/well were plated into 96-well plates, and cell proliferation was monitored using the CellTiterGlo assay to measure ATP usage. LK2-EREB2-5 cells with estrogen (□) and without estrogen (▪), LK2-EBNA2 cells with estrogen (▵) and without estrogen (▴), and LK2-EBNA2ΔCR4 cells with estrogen (○) and without estrogen (•) are shown. Data shown are the means for three assays, with standard deviations provided.
FIG. 2.
FIG. 2.
EBNA2ΔCR4 is transcriptionally competent in supporting LMP1 expression and Cp promoter activity. (A) Upper panel, Western blot probed with anti-EBNA2 PE2 monoclonal antibody (DAKO). ER-EBNA2 is expressed in EREB2-5 cells transduced with LK2. ER-EBNA2 plus EBNA2 are expressed in LK2-EBNA2- and LK2-EBNA2ΔCR4-transduced cells. Middle panel, Western blot showing loss of ER-EBNA2 expression in LK2-EBNA2 and LK2-EBNA2ΔCR4 cells grown continuously in the absence of estrogen. LK2-EREB2-5 cells maintained in estrogen-containing medium retain ER-EBNA2 expression. Lower panel, the membrane shown in the middle panel was stripped and reprobed with anti-LMP1 S12 monoclonal antibody (32a). LK2-EBNA2ΔCR4 cells showed no deficit in the ability to mediate LMP1 expression. (B) Transient expression assay in which HeLa cells were cotransfected with the EBV Cp promoter reporter 4× Cp-CAT and either control vector or an expression plasmid for EBNA2, the EBNA2 mutant (EBNA2WW), or EBNA2ΔCR4. EBNA2ΔCR4 was as effective as EBNA2 in activating expression of 4× Cp-CAT.
FIG. 3.
FIG. 3.
EBNA2 CR4 is necessary for resistance to etoposide- and 5-FU-induced cell death but not TNF-α-induced cell death. Estrogen-independent LK2-EBNA2ΔCR4 and LK2-EBNA2 cells were treated with (A, B) etoposide (10 μg/ml), (C, D) 5-FU (25 ng/ml), or (E, F) TNF-α (5 ng/ml) plus cycloheximide (10 μg/ml; CalBiochem) for 20 h. (G) EREB2-5 cells were grown in the absence of estrogen for 5 days before treatment with TNF-α plus cycloheximide. Apoptosis was measured by annexin V-phycoerythrin binding. The data shown are representative of three independent experiments. Drug-treated cells, shaded profile; untreated cells, open profile.
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References

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