Fig 5. EV71 2Apro inhibits the biosynthesis of Herp and VIMP.
(A) RD cells were mock-infected (−) or infected (+) with EV71 (MOI = 10) for 9 h and then treated with MG132 (50 μM), Tg (300 nM), Tg plus MG132, Tun (10 μg/ml), Tun plus MG132 for an additional 6 h. Then, the cells were harvested and analyzed by western blotting with antibodies against Herp, EV71 VP1, and actin. The lower panel graph shows the quantification of Herp. The data are presented as means ± SD of two independent experiments. (B) RD cells were mock-infected (−) or infected (+) with EV71 (MOI = 10) for 9 h and then treated with Tg (300 nM) or Tun (10 μg/ml) for 6 h to induce Herp expression. Then, the mRNA expression levels of Herp were evaluated by quantitative real-time PCR. The data are presented as means ± SD of two independent experiments. NS, non-significant, P≥0.05; ** P<0.01. (C) BSRT7 cells were transfected with pcDNA3.1-EGFP, pcDNA3.1-IRES-2A, or pcDNA3.1-IRES-2A(C110A). At 36 h post-transfection, cells were treated with Tg (300 nM), Tg plus MG132 (50 μM), Tun (10 μg/ml), or Tun plus MG132 for 6 h. Then, cell lysates were analyzed by western blotting with antibodies against Herp, eIF4GI, V5, GFP, and actin. eIF4GI was included as a positive control of protease activity of 2Apro, and arrows indicate the cleavage fragments (CF) of eIF4GI. (D) BSRT7 cells were transfected with increasing doses of pcDNA3.1-EGFP plasmid or pcDNA3.1-IRES-2A plasmid (1–3 μg). At 36 h post-transfection, the cells were harvested, RNA was extracted, and quantitative real-time PCR was used to analyze VIMP mRNA expression. The data are presented as means ± SD of three independent experiments. NS, non-significant, P≥0.05. (E) BSRT7 cells were transfected with increasing doses of pcDNA3.1-EGFP, pcDNA3.1-IRES-2A, or pcDNA3.1-IRES-2A(C110A) plasmids (1–3 μg). At 36 h post-transfection, the cells were harvested and cell lysates were analyzed by western blotting with antibodies against VIMP, c-MYC, p97, eIF4GI, GFP, V5, and actin.