Figure 4.

A3G competitively binds to the EV71 5′UTR with PCBP1. (A and B) A3G and PCBP1 expression was detected by immunoblotting analysis. pcDNA3.1, A3G-HA, A3G C291S-HA or PCBP1-HA was co-transfected with the 5′UTR expression vector into HEK293T cells. Cell lysates were prepared at 48 h post-transfection. Part of the cell lysates were immunoprecipitated with anti-HA agarose beads. (B) Binding capacity of A3G or PCBP1 to the EV71 5′UTR. The results are the means with SD from at least three independent experiments. (C) The interaction between A3G or PCBP1 with the 5′UTR of EV71 according to RNA pull-down assay. (D–F) Competitive binding assay using immunoprecipitation. HEK293T cells were transfected with increasing doses of A3G-myc and PCBP1-HA plus the 5′UTR. At 48 h post-transfection, half of the cells were harvested and immunoprecipitated with anti-HA agarose beads, and the other cells were precipitated with anti-myc agarose beads. The cell lysates and immunoprecipitated products were analysed by immunoblotting (D) and RT-qPCR analyses. (E) 5′UTR RNA input in cell lysates. GAPDH was used as a control. (F) Increasing amounts of A3G disrupted the interaction of PCBP1 with the EV71 5′UTR. The binding between the 5′UTR and PCBP1 in the absence of A3G was set as 100%. The results are the means with SD from at least three independent experiments. The asterisks indicate statistically significant differences between groups as assessed by Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). (G) Increasing amounts of A3G decreased the interaction of PCBP1 with the 5′UTR of EV71 according to RNA pull-down assay.