Fig 2: 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.

Fig 3: A3G inhibits EV71 5′UTR activity. (A) Bicistronic plasmid construction. (B–D) pcDNA3.1, A3G or A3G C291S plus the pIRIGF negative vector or bicistronic pIRIGF-5′UTR expression plasmid were co-transfected into HEK293T cells, which were harvested at 48 h post-transfection. (B) A3G and A3G C291S expression was detected by immunoblotting analysis. (C) Effects of A3G and A3G C291S on luciferase mRNA levels according to RT-qPCR analysis. GAPDH was used as a control. mRNA levels of luciferase downstream of CMV in the absence of A3G were set as 100%. (D) Effects of A3G and A3G C291S on luciferase activity. Luciferase activity downstream of CMV in the absence of A3G was set as 100%. (C and D) 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 < 0.001).

Fig 4: The functional domain of EV71 2C is required for A3G degradation. (A) EV71 2C mutant construction. (B and C) The amino-terminal domain but not the carboxy-terminal domain of EV71 2C is required for 2C-mediated degradation. HEK293T cells were co-transfected as indicated and then harvested for immunoblotting analysis at 48 h post-transfection. (D) Truncated mutants in the amino-terminal domain of EV712C were constructed. (E) Amino acids 26–40 were required for the 2C-mediated degradation of A3G.

Fig 5: A3G and the A3G C291S mutant both inhibit EV71 replication in HEK293T cells infected with EV71 virus. HEK293T cells were transfected with pcDNA3.1, A3G-myc or A3G C291S-myc and then infected with EV71 virus at an MOI of 1.0 at 24 h post-transfection. The cells and supernatants were harvested at 24 h, 48 h and 72 h post-infection. (A) A3G and viral VP1 levels in the cells and supernatants were detected by immunoblotting analyses using anti-VP1, anti-myc and anti-tubulin antibodies. The supernatants from transfected HEK293T cells were concentrated using 25% sucrose prior to immunoblotting analysis. (B) EV71 RNA levels in cells were detected by RT-qPCR. GAPDH was used as a control. EV71 RNA levels of cells transfected with pcDNA3.1 for 24 h were set as 100%. (C) Viral titres in the supernatants were determined by the cytopathic effect method. The results are the means with SD from three independent experiments. The asterisks indicate statistically significant differences between groups as assessed by Student's t-test (**P < 0.01). The endogenous expression levels of PPIA or ElonginC in HEK293T cells (D) and A3C in Jurkat cells (E) were detected by immunoblotting analyses at 72 h post-infection.