Inhibition of Epstein-Barr virus (EBV) reactivation by short interfering RNAs targeting p38 mitogen-activated protein kinase or c-myc in EBV-positive epithelial cells

Abstract

Latent Epstein-Barr virus (EBV) is reactivated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in EBV-infected cells. In this study, we found that TPA up-regulated phosphorylation of p38, a mitogen-activated protein kinase, and activated c-myc mRNA in EBV-positive epithelial GT38 cells. The EBV immediate-early gene BZLF1 mRNA and its product ZEBRA protein were induced following TPA treatment. Protein kinase C inhibitors, 1-(5-isoquinolinesulphonyl)-2, 5-dimethylpiperazine (H7) and staurosporine, inhibited the induction of p38 phosphorylation and the activation of c-Myc by TPA. The p38 inhibitor SB203580 blocked both p38 phosphorylation and ZEBRA expression by TPA. Pretreatment of GT38 cells with the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine inhibited p38 phosphorylation and c-Myc activation by TPA, suggesting that NO may inhibit EBV reactivation via both p38 and c-Myc. By using short interfering RNA (siRNA) targeting either p38 or c-myc, we found that p38 or c-myc siRNA specifically inhibited expression of the respective gene and also suppressed the induction of ZEBRA and EBV early antigen. The interferon (IFN)-responsive gene expression tests ruled out the possibility that the antiviral effect of siRNA is dependent on IFN. Our present study demonstrates for the first time that either p38 or c-myc siRNA can efficiently inhibit TPA-induced EBV reactivation in GT38 cells, indicating that p38- and/or c-myc-associated signaling pathways may play critical roles in the disruption of EBV latency by TPA.

FIG. 1.

TPA induces c-myc and p38 gene expression and EBV reactivation. GT38 cells were incubated with TPA (20 ng/ml). (A) Total RNAs were isolated from the cells at indicated times, and the expression levels of BZLF1, BRLF1, and c-myc genes were analyzed by Northern blotting. GAPDH was analyzed as an internal control standard. (B) Cell lysates were isolated at indicated times, and the phosphorylation of p38, total p38 (t-p38), c-Myc, and ZEBRA were analyzed by Western blotting with anti-phospho-p38, anti-p38, anti-c-Myc, and anti-ZEBRA antibodies. Total p38 and β-actin were analyzed as internal control standards. The relative densitometric units of BRLF1, BZLF1, and c-myc to GAPDH (panel A) and of phospho-p38 to total p38 and c-Myc and ZEBRA to β-actin (panel B) were quantified by a phosphorimage system. The data represent three independent experiments, and the standard deviations are shown.

FIG. 2.

Inhibitors of PKC and p38 specifically suppress the TPA-mediated expression levels of c-myc, p38, and ZEBRA. (A) GT38 cells were pretreated with a PKC inhibitor H7 (100 μM) or staurosporine (St; 0.1 μM) for 1 h, and then exposed to TPA (20 ng/ml) for 4 h. Total RNAs were extracted and analyzed for c-myc mRNA expression by Northern blotting. After stripping, the membrane was reprobed with GAPDH. (B) GT38 cells were pretreated with H7 or staurosporine (St) or SB203580 (SB; 10 μM) for 1 h and then treated with TPA. Cell lysates were extracted for p38, c-Myc, and ZEBRA at 0.5, 4, and 48 h, respectively, after TPA treatment. The phosphorylation of p38, total p38 (t-p38), c-Myc, phospho-c-Myc, ZEBRA, and β-actin were analyzed by Western blotting with anti-phospho-p38, anti-p38, anti-c-Myc, anti-phospho-c-Myc, anti-ZEBRA, or anti-β-actin antibodies. The relative densitometric units of c-myc to GAPDH (panel A) and of phospho-p38 to total p38, as well as c-Myc, phospho-c-Myc, and ZEBRA to β-actin (panel B) were quantified with a phosphorimage system. The data represent three independent experiments and the standard deviations are shown.

FIG. 3.

TPA-induced phosphorylation of p38 and activation of c-Myc were inhibited by the NO donor SNAP. GT38 cells were pretreated with SNAP at indicated concentrations for 1 h prior to TPA (20 ng/ml) treatment. Cell lysates were prepared for phosphorylation of p38 and total p38 (t-p38) and of c-Myc at 0.5 and 4 h, respectively, after TPA treatment. Cell lysates were separated by SDS-PAGE and blotted onto membranes. Phosphorylated p38, p38, and c-Myc were detected by Western blotting with anti-phospho-p38, anti-p38, anti-c-Myc or anti-β-actin antibodies. The p38 and β-actin were used as internal standards. The relative densitometric units of phosphorylation of p38 and activation of c-Myc were determined to p38 and β-actin signals, respectively. The data represent three independent experiments and the standard deviations are shown.

FIG. 4.

siRNAs targeting p38 and c-myc specifically inhibit the expression of respective genes and ZEBRA expression. (A) GT38 cells were transfected with p38 siRNA (0.1 μM) or scrambled siRNA (scr) and incubated for 48 and 72 h or for 72 h and then treated with TPA for 30 min for phosphorylated p38 and total p38 (t-p38) and 48 h for ZEBRA. The cell extracts were analyzed for phosphorylation of p38, total p38, ZEBRA, and β-actin by Western blotting. (B) GT38 cells were transfected with siRNA (0.1 μM) targeted to c-myc for 48 or 72 h or with scrambled (scr) siRNA for 72 h and then treated with TPA (20 ng/ml) for 4 or 48 h for the expression analysis of c-Myc and ZEBRA, respectively. Cell lysates were harvested from siRNA-transfected or untransfected GT38 cells at indicated times posttransfection. C-Myc and ZEBRA expression were analyzed by Western blotting by using anti-c-Myc, anti-ZEBRA, and anti-β-actin antibodies. The signals of phosphor-p38, t-p38, and ZEBRA (panel A) and of c-Myc and ZEBRA (panel B) were quantified by a phosphorimage system. The relative protein level was measured as ratio of β-actin protein. The data represent three independent experiments, and the standard deviations are shown.

FIG. 5.

p38 or c-myc siRNA blocks EA induction by TPA. GT38 cells were transfected with siRNA (0.1 μM) targeting p38 and c-myc or with scrambled (scr) siRNA. At 48 h posttransfection, cells were treated with TPA (20 ng/ml) for 72 h. EA-positive cells were determined by indirect immunofluorescence. The data shown represent three independent experiments. Bars represent standard deviations. (*, P < 0.01 versus the TPA-positive and siRNA-negative cells).

FIG. 6.

Effects of siRNAs targeted to p38 and c-myc on the 2,5 OAS induction and cell growth. (A) GT38 cells were transfected with p38, c-myc, or scrambled (scr) siRNA. At 48 h posttransfection, cells were treated with TPA (20 ng/ml) for 48 h. As the positive control of 2,5 OAS, GT38 cells were treated with IFN (300 U/ml) for 6 h. The expression levels of 2,5 OAS1 and 2,5 OAS2 were determined by RT-PCR. β-tubulin mRNA was amplified from the same cDNA as an internal standard. (B) GT38 cells were plated in 96-well plates and incubated for 24 h and then transfected with siRNA duplexes targeted to c-myc or p38 or scrambled (scr) siRNA. Cell growth was analyzed by using MTT assays.