Fig 2: EV71 2C antagonizes A3G via the autophagy–lysosome degradation pathway but not the proteasome pathway. (A) 2C reduced the expression of A3G. A3G-HA plus VR1012 (negative control) or the indicated EV71 non-structural proteins were co-transfected into H9 cells. The cells were harvested for immunoblotting analysis at 48 h post-transfection. (B) The MG132 inhibitor could not rescue EV71 2C-mediated A3G degradation. The cells were treated for another 12 h with 10 μM DMSO or MG132 prior to harvest for immunoblotting analysis at 36 h post-transfection. (C) An autophagy inhibitor could rescue EV71 2C-mediated A3G degradation. The cells were treated for another 12 h with 3 μM DMSO, 3 μM thapsigargin or 10 nM Baf-A1 prior to harvest for immunoblotting analysis at 36 h post-transfection. (D) EV71 2C interacted with A3G according to immunoprecipitation assay. HIV-1 Vif-HA was used as a positive control. The cells were treated for another 12 h with 10 μM DMSO, 10 μM MG132 or 10 nM Baf-A1 to avoid A3G degradation as indicated prior to harvest. (E) A3G was ubiquitinated by EV71 2C. (F) Co-localization of EV71 2C and A3G in HeLa cells. 2C formed autophagic puncta and co-localized with A3G. (G) Co-localization of A3G and p62 in the presence or absence of EV71 2C. (F and G) Images were taken under a Zeiss LZM710 confocal microscope.

Fig 3: H9 cells expressing A3G have lower EV71 replication capacity than Jurkat cells without A3G expression. (A and B) EV71 replication was lower in H9 cells than in Jurkat cells. H9 and Jurkat cells were infected with DMEM or EV71 virus at an MOI of 1.0. The infected cells were harvested at the indicated time points post-infection. (A) A3G and viral VP1 levels in cells were detected by immunoblotting analyses using anti-VP1, anti-A3G and anti-tubulin antibodies. (B) EV71 RNA levels were lower in H9 cells than in Jurkat cells according to RT-qPCR detection. EV71 RNA levels of Jurkat cells infected with EV71 for 24 h were set as 100%. (C–F) Silencing A3G in H9 cells enhanced EV7 replication. H9 cells stably expressing A3G shRNA were established. A3G protein (C) and mRNA (D) levels at different time points are shown. (E) EV71 RNA levels were higher in A3G knockdown H9 cells than in negative control pLKO.1 cells at all time points. EV71 RNA levels of negative control pLKO.1 cells infected with EV71 for 48 h were set as 100%. (F) Cytotoxicity induced by EV71 in A3G knockdown H9 cells and control pLKO.1 cells was detected by CCK8 assays. Uninfected H9 cells at 0 h were set as 100%. (B, D–F) 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 < 0.05, **P < 0.01, ***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.