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. 2005 Jul;79(13):8655-60.
doi: 10.1128/JVI.79.13.8655-8660.2005.

Epstein-Barr virus (EBV) latent membrane protein 2A regulates B-cell receptor-induced apoptosis and EBV reactivation through tyrosine phosphorylation

Makoto Fukuda  1 , Richard Longnecker
Affiliations

Epstein-Barr virus (EBV) latent membrane protein 2A regulates B-cell receptor-induced apoptosis and EBV reactivation through tyrosine phosphorylation

Makoto Fukuda et al. J Virol. 2005 Jul.

Abstract

Epstein-Barr virus (EBV) is a human herpesvirus that establishes a lifelong latent infection of B cells. Within the immune system, apoptosis is a central mechanism in normal lymphocyte homeostasis both during early lymphocyte development and in response to antigenic stimuli. In this study, we found that latent membrane protein 2A (LMP2A) inhibited B-cell receptor (BCR)-induced apoptosis in Burkitt's lymphoma cell lines. Genistein, a specific inhibitor of tyrosine-specific protein kinases, blocked BCR-induced apoptosis and EBV reactivation in the cells. These findings indicate that LMP2A blocks BCR-induced cell apoptosis and EBV reactivation through the inhibition of activation of tyrosine kinases by BCR cross-linking.

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Figures

FIG. 1.
FIG. 1.
LMP2A inhibits BCR-induced DNA fragmentation and cleavage of PARP in Ramos cells. (A) Cells were seeded at 3 × 105 cells/ml, and cells were treated without (control) or with 35 μg/ml anti-IgM antibody (αIgAb) for 24 h or 48 h. Cells were analyzed for DNA content by propidium iodide (PI) staining and flow cytometry. Gates employed to ascertain cell cycle distribution and the percentage of cells with a sub-G1 (<G1) and G2/M DNA content are shown. These data are representative of three experiments. (B) PARP cleavage was analyzed by immunoblotting with a specific anti-PARP antibody. The full-length 113-kDa and 89-kDa cleaved PARP proteins are indicated. The amount of protein loaded in each lane was assessed by rehybridization of the filter with a specific antibody for human GAPDH. P, parental; V, vector control; 2A, LMP2A expressing. (C) DNA fragmentation and (D) evaluation of cleavage of PARP. Cells (3 × 105/ml) were preincubated for 1 h with or without zVAD-fmk (50 μM), and cells were then treated with 35 μg/ml anti-IgM antibody. After 24 h of incubation, cell cycles and PARP cleavage were analyzed as described in the legend to panel B. The amount of protein loaded in each lane was assessed by rehybridization of the filter with a specific antibody for human GAPDH.
FIG. 2.
FIG. 2.
LMP2A or genistein inhibits BCR-induced tyrosine phosphorylation, DNA fragmentation, and cleavage PARP in Ramos cells. (A) Parental (P), vector control (V), and LMP2A-expressing (2A) Ramos cells (1 × 106/ml) were treated without (control [−]) or in the presence of 35 μg/ml anti-IgM antibody (αIgM Ab) for the indicated times (1, 5, or 10 min). (B) Cells (1 × 106/ml) were preincubated for 30 min with various concentrations of genistein, and then cells were treated with 35 μg/ml anti-IgM antibody for 10 min. Equal amounts of protein from the respective cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The levels of expression of phosphorylated tyrosine were determined by immunoblotting. (C) DNA fragmentation and (D) evaluation of cleavage of PARP. Cells (3 × 105/ml) were preincubated for 30 min with or without genistein, and cells were then treated with 35 μg/ml anti-IgM antibody. After 24 h of incubation, cell cycles and PARP cleavage were analyzed as described in the legend to Fig. 1. The amount of protein loaded in each lane was assessed by rehybridization of the filter with a specific antibody for human GAPDH.
FIG. 3.
FIG. 3.
LMP2A or genistein blocks BCR-induced DNA fragmentation, cleavage of PARP, and EBV reactivation. (A) DNA fragmentation and evaluation of cleavage of PARP. Parental (P), vector control (V), and LMP2A-expressing (2A) Akata cells (5 × 105/ml) were preincubated for 30 min with or without genistein, and cells were then treated with 40 μg/ml anti-IgG antibody (αIgG Ab). After 24 h of incubation, cell cycle analyses and PARP cleavage were analyzed as described in the legend to Fig. 1. (B) EBV reactivation. Cells (5 × 105/ml) were preincubated for 30 min with or without genistein (G; 50 μg/ml), and cells were then treated with 40 μg/ml anti-IgG antibody. After 24 h of incubation, the expression of ZEBRA (38 kDa) and EA (52 kDa and 55 kDa) was analyzed by immunoblotting with a specific anti-ZEBRA antibody or with human EBV serum reactive with EBV early antigens. The amount of protein loaded in each lane was assessed by rehybridization of the filter with a specific antibody for human GAPDH. (C) EBV reactivation, (D) DNA fragmentation, and (E) cleavage of PARP with zVAD-fmk treatment. Cells (5 × 105/ml) were preincubated for 1 h with or without zVAD-fmk (50 μM), and cells were then treated with 40 μg/ml anti-IgG antibody. After 24 h of incubation, cell cycles and the expression of ZEBRA were analyzed as described in the legends to Fig. 1 and panel B. The amount of proteinloaded in each lane was assessed by rehybridization of the filter with a specific antibody for human GAPDH. (F) Tyrosine phosphorylation with zVAD-fmk treatment. Cells (1 × 106/ml) were preincubated for 1 h with or without zVAD-fmk (50 μM) followed by treatment with 35 μg/ml anti-IgM antibody for 10 min. Equal amounts of protein from the respective cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The levels of expression of phosphorylated tyrosine was determined by immunoblotting.
FIG. 4.
FIG. 4.
PMA and calcium ionophore A23187 induce apoptosis and EBV reactivation in LMP2A-expressing Ramos and Akata cells. (A) Parental (P), vector control (V), and LMP2A-expressing (2A) Ramos and Akata cells (1 × 106/ml) were treated without (control [−]) or with of 35 μg/ml anti-IgM antibody or 40 μg/ml anti-IgG antibody for the indicated times (1, 5, or 10 min). Equal amounts of protein from the respective cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ERK1/2 phosphorylation was detected with anti-phospho MAPK antibody. Upper and lower arrows indicate ERK1 and ERK2, respectively. The lower panels show equal loading of proteins and the expression of total ERK (ERK1/2). (B) DNA fragmentation, evaluation of cleavage of PARP, and (C) EBV reactivation. Parental (P), vector control (V), and LMP2A (2A)-expressing Ramos (3 × 105/ml) and Akata (5 × 105/ml) cells were treated with 20 ng/ml PMA and 7.5 μM A23187. After 24 h of incubation, cell cycles, PARP cleavage, and the expression of ZEBRA and EA were analyzed as described in the legends to Fig. 1 and Fig. 3.
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