Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

Figure 1.. 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…

Figure 2.. 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)…

Figure 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).

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.…

Figure 5.. The RNA-binding property of A3G is required for its binding to the EV71 5′UTR and EV71 inhibition. (A) The amino-terminal domain of A3G alone could inhibit EV71 replication, but the carboxy-terminal could not. HEK293T cells were transfected with pcDNA3.1(–), A3G or the indicated mutants. At 24 h post-transfection, HEK293T cells were infected with EV71 at an MOI of 0.1. At 72 h post-infection, the cells were harvested and loaded for immunoblotting analyses using anti-VP1, anti-HA and anti-tubulin antibodies. The amino-terminal of A3G alone could inhibit EV71 replication. (B) Immunoprecipitation assay. HEK293T cells were transfected with A3G-HA or the indicated mutants plus the 5′UTR expression vector. At 48 h post-transfection, the cells were harvested and immunoprecipitated with anti-HA agarose beads. The cell lysates and immunoprecipitated products were analysed by immunoblotting. (C) The amino-terminal domain of A3G lost the ability to interact with the 5′UTR according to RT-qPCR analysis. GAPDH was used as a control. (D) EMSA of the EV71 5′UTR and A3G or its mutants. (E) A3G mutants L123A, Y124A and W127A could not inhibit EV71 replication. The assay was performed as described…

Figure 6.. (A) The secondary structure of the 5′UTR was predicted by MFold. (B–D) The interactions between A3G and the 5′UTR truncated mutants. A3G or VR1012 and the WT 5′UTR or indicated 5′UTR truncations were transfected into HEK293T cells; then, the cells were harvested for IP and RT-PCR analysis at 48 h post-transfection. (B) A3G protein levels in the cell lysates and co-IP elutes were confirmed by immunoblotting analysis. (C) 5′UTR RNA input in the cell lysates was detected by RT-qPCR. (D) Loop I and loop II in the EV71 5′UTR maintained binding ability with A3G according to RT-qPCR detection. The RNA level of the WT 5′UTR binding to A3G was set as 100%. 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). (E) EMSA of the EV71 5′UTR or its truncated mutants with A3G.

Figure 7.. 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)…

Figure 8.. 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.