Fig 2: EGCG inhibits EBV lytic replication by suppressing LMP1-mediated MAPK signal axes in AGS-EBV and B95.8 cells. AGS-EBV and B95.8 cells were starved in 0.1% FBS HAM’s/F-12 or RPMI-1640 medium for 24 h and then treated with different concentrations of EGCG as indicated or vehicle [dimethyl sulfoxide (DMSO)] for 24 h. (A, B) Western blotting analysis was performed to determine LMP1, total and phosphorylated ERKs, p38, and JNKs, total and phosphorylated p53, and total and phosphorylated c-Jun. (C) Effects of EGCG on the expression of Zta and Ea-D. ß-Actin was used as an internal control. (D) Reverse transcription (RT)-qPCR quantification of mRNA expression levels for BZLF1 and BMRF1 genes. (E) Flow cytometry was performed to analyze the effects of EGCG on the numbers of Zta- or Ea-D-positive cells. Incubation with vehicle but no primary antibody followed by incubation with the secondary antibody was used as a negative control. (F) The changes in EBV genome copies were analyzed using qPCR. The levels of EBV DNA copy number from the control group were set to 1. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group.

Fig 3: LMP1 enhances the ability of wt-p53 to induce expression of EBV lytic proteins through ERKs, p38, and JNKs in AGSEBV cells. (A) AGS-EBV cells were transfected with a plasmid containing pSG5-LMP1 or a control pSG5 vector for 48 h. Western blotting analysis was performed to detect the levels of LMP1, total MAPKs and p53, and phosphorylated MAPKs and p53. (B–D) Western blotting analysis for EBV lytic proteins and phosphorylated p53 in AGS-EBV cells pretreated with PD98059, SB202190, or Sp600125 for 24 h at the concentrations indicated. (E) AGS-EBV cells were transfected with plasmid expressing LMP1 or empty vector for 48 h. Coimmunoprecipitation (Co-IP) assay using anti-p53 antibody was performed to analyze the interactions of p53 with Sp1 and SMAD2/3. The resulting immunocomplexes were recovered and analyzed by Western blotting analysis. ß-Actin was used as an internal control.

Fig 4: Schematic illustrating EGCG inhibition of EBV lytic replication by directly targeting LMP1-mediated MAPK signal axes. LMP1 recruits TRAFs to constitutively activate MAPKs, including ERKs, p38, and JNKs, promoting EBV lytic replication. LMP1-induced phosphorylation of wt-p53 at Ser15, Ser20, and Ser392 through MAPKs can enhance the ability of p53 to activate Zp by directly binding to Zp as well as recruiting transactivators Sp1 and SMAD2. When p53 is in a mutated state, phosphorylation of c-Jun at Ser63 and Ser73 induced by LMP1 through JNKs is involved in regulating Zta expression. Polyphenol compound EGCG can directly target LMP1 to block the MAPKs/wt-p53 and JNKs/c-Jun signal axes, effectively inhibiting EBV lytic replication.

Fig 5: Mitogen-activated protein kinases (MAPKs) are required for EBV lytic replication induced by LMP1. (A) AGS-EBV and B95.8 cells were transfected with a plasmid containing pSG5-LMP1 or a control pSG5 vector. Western blotting analysis for LMP1, total and phosphorylated extracellular signal-regulated kinases (ERKs), p38, and c-Jun NH2-terminal kinases (JNKs), and EBV lytic proteins Zta and Ea-D were performed. AGS-EBV and B95.8 cells were pretreated with the MAPK-specific inhibitors, including PD98059 (B), SB202190 (C), and Sp600125 (D), at the concentrations as indicated for 24 h, respectively. Western blotting analysis was performed to detect total and phosphorylated ERKs, p38, and JNKs, and EBV lytic proteins Zta and Ea-D. AGS-EBV and B95.8 cells were transfected with the plasmid expressing LMP1 for 4 h prior to incubation with the MAPK-specific inhibitors alone or in combination, and then these cells were continuously cultured for 48 h. (E) The levels of total and phosphorylated MAPKs, lytic proteins, and (F) EBV DNA copy numbers were analyzed using Western blotting and qPCR, respectively. For each experiment, EBV DNA copy number levels from the control group were set to 1. ß-Actin was used as an internal control. *p < 0.05, **p < 0.01 versus control group.