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. 2007 Jul;81(14):7598-607.
doi: 10.1128/JVI.02435-06. Epub 2007 May 9.

Truncated form of the Epstein-Barr virus protein EBNA-LP protects against caspase-dependent apoptosis by inhibiting protein phosphatase 2A

Julie Garibal  1 , Emilie Hollville, Andrew I Bell, Gemma L Kelly, Benjamin Renouf, Yasushi Kawaguchi, Alan B Rickinson, Joëlle Wiels
Affiliations

Truncated form of the Epstein-Barr virus protein EBNA-LP protects against caspase-dependent apoptosis by inhibiting protein phosphatase 2A

Julie Garibal et al. J Virol. 2007 Jul.

Abstract

The Epstein-Barr virus (EBV)-encoded leader protein, EBNA-LP, strongly activates the EBNA2-mediated transcriptional activation of cellular and viral genes and is therefore important for EBV-induced B-cell transformation. However, a truncated form of EBNA-LP is produced in cells infected with variant EBV strains lacking EBNA2 due to a genetic deletion. The function of this truncated form is unknown. We show here that some Burkitt's lymphoma cells harboring defective EBV strains are specifically resistant to the caspase-dependent apoptosis induced by verotoxin 1 (VT-1) or staurosporine. These cells produced low-molecular-weight Y1Y2-truncated isoforms of EBNA-LP, which were partly localized in the cytoplasm. The transfection of sensitive cells with constructs encoding truncated EBNA-LP isoforms, but not full-length EBNA-LP, induced resistance to caspase-mediated apoptosis. Furthermore, VT-1 induced protein phosphatase 2A (PP2A) activation in sensitive cells but not in resistant cells, in which the truncated EBNA-LP interacted with this protein. Thus, the resistance to apoptosis observed in cells harboring defective EBV strains most probably results from the inactivation of PP2A via interactions with low-molecular-weight Y1Y2-truncated EBNA-LP isoforms.

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Figures

FIG. 1.
FIG. 1.
VT-1 and staurosporine induce caspase-dependent apoptotic cell death in some, but not all, BL cell lines. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.
FIG. 2.
FIG. 2.
Infection with the P3HR1 strain of EBV protects BL cells against VT-1- and staurosporine-induced apoptosis. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.
FIG. 3.
FIG. 3.
Levels of expression of various EBV latent proteins and subcellular distributions of EBNA-LP in various BL cell lines and derived cell clones. (A) Cell pellets were lysed and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb, CS.1-4 anti-LMP-1 MAb, 1H4 anti-EBNA1 MAb, PE2 anti-EBNA2 MAb, or an anti-β-actin MAb (as a control for protein loading). (B and C) Total lysates (T) were fractionated into cytoplasmic (C) and nuclear (N) extracts. Equal amounts of each fraction (20 μg protein) were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb. We checked that fractionation was complete by probing the membranes with MAbs recognizing a nuclear protein (topoisomerase II) and a cytoplasmic protein (IκBα). Molecular sizes are indicated to the right.
FIG. 4.
FIG. 4.
Production levels, subcellular distributions, and effects on apoptosis of EBNA-LP after transient transfection. (A) MCF7 cells were transfected with 3 μg of various EBNA-LP expression vectors and grown for 48 h. Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb or an anti-β-actin MAb (as a control for protein loading). Molecular sizes are indicated to the right. (B) MCF7 cells were transfected with 3 μg of various EBNA-LP expression vectors and grown on glass coverslips; 48 h after transfection, the cells were stained with 4D3 anti-EBNA-LP MAb and GAM-Alexa 488. (C) MCF7 cells were transfected with 3 μg of various EBNA-LP expression vectors; 48 h after transfection, the cells were treated with staurosporine (2.5 μM) for 6 h and stained with PI. The levels of apoptosis were assessed by determining the proportions of cells with sub-G1 DNA content by flow cytometry. The percentages of apoptosis inhibition were determined by comparison with cells transfected with the pSG5 vector. Error bars show the standard deviations.
FIG. 5.
FIG. 5.
Effect of stable transfection of EBNA-LP on apoptosis. Ramos cells were cotransfected with EBNA-LP expression vectors and the pSG5(NeoR) vector, which carries the neomycin resistance gene. The cells were grown in selection medium (complete RPMI supplemented with 16 mg/ml neomycin) for 4 weeks, and the clones were then tested. (A) Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb. Molecular sizes are indicated to the right. (B) Stable transfectants were treated with VT-1 (5 ng/ml) for 16 h, labeled with annexin V-FITC and PI, and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. The percentages of apoptosis inhibition were determined by comparison with cells transfected with the pSG5 vector. Error bars shown the standard deviations.
FIG. 6.
FIG. 6.
PP2A is involved in VT-1-induced apoptosis. (A) Ramos cells were incubated with okadaic acid (50 nM) or with vehicle (DMSO) or without any treatment (−) for 1 h before being treated with VT-1 for 4 h. The cells were labeled with annexin V-FITC and PI and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. (B) VT-1-sensitive or -resistant cells were incubated with or without VT-1 (5 ng/ml) for various periods of time and lysed. The lysates were immunoprecipitated with an anti-PP2A Ab, and the levels of PP2A activity were determined by measuring the release of phosphate from a phosphopeptide substrate in a colorimetric assay. The increases in the PP2A activity levels of treated samples were determined with respect to the levels in untreated samples. Error bars indicate the standard deviations.
FIG. 7.
FIG. 7.
PP2A interacts with EBNA-LP in cells resistant to VT-1-induced apoptosis. Lysates obtained from various VT-1-sensitive or -resistant cells were subjected to immunoprecipitation (IP) with 4D3 anti-EBNA-LP MAb or irrelevant Abs (Irr). Western blot analysis was then performed with an anti-PP2Ac Ab or 4D3 anti-EBNA-LP MAb. As a control for protein levels before IP, a portion of cell lysate (Input) corresponding to 15% of the input for IP was also included in the Western blot analysis. Molecular sizes are indicated to the right.
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