Enterovirus 71 Activates GADD34 via Precursor 3CD to Promote IRES-Mediated Viral Translation

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  FIG 1. GADD34 identified by Riboseq screen to be upregulated during EV71 infection. (A) Riboseq and RNA-Seq screening processes. RD cells were infected with 40 MOI EV71 for 3.5 h and 6.25 h, compared with mock cells, to establish ribosome-protected fragment (RPF) and total mRNA libraries. (B) High-throughput screening results. The RPF and mRNA changes of GADD34 in the two biological replicates. (C) RD cells were infected with 10 MOI EV71, and the cells were harvested at different times after infection. GADD34 mRNA was detected by RT-qPCR. (D) After RD cells were infected with 5 MOI EV71 for a gradient time, GADD34 was detected by Western blotting. (E) RD cells were infected with EV71 with a gradient of MOI for 8 h, and the expression of GADD34 protein was detected by Western blotting. (F) HT-29 cells were infected with 5 MOI EV71, and the cells were harvested at different times after infection. GADD34 mRNA was detected by RT-qPCR. (G) After HT-29 cells were infected with 5 MOI EV71 for a gradient time, GADD34 was detected by Western blotting. Values were the means plus standard errors of the means (SEM) (error bars) from two individual experiments. Values were statistically evaluated…

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  FIG 2. EV71 non-structural precursor protein 3CD upregulates GADD34. (A) Schematic diagram of EV71 genome structure. (B) Screen of the viral components that activate GADD34 protein. pCE-puro-3×Flag-3A, 3AB, 3C, 3D, and 3CD were transfected into HeLa cells, and GADD34 protein expression was detected by Western blotting after 48 h. (C) Schematic diagram of constructing 3CD mutation sites. (D) HeLa cells were transfected with EV71 3CD wild-type and 3CD nuclear localization signal mutation R205AK207A, protease activity mutation E71A, and RNA binding mutation K82QR84Q, GADD34 was detected by Western blotting 48 h after transfection. (E) The mutants L630D and K12L of poliovirus 3CD interfere with different stages of Arf-PI4P activation. (F) R12, Y441, and I631 of EV71 3CD are conserved in enterovirus. (G) HeLa cells were transfected with EV71 3CD wild-type and 3CD mutant Y441S, R12L, or I631D, GADD34 was detected by Western blotting 48 h after transfection. (H) Plasmids infectious clone pSVA-EV71, pSVA-EV71-3CD-Y441S, pSVA-EV71-3CD-I631D, or pSVA-EV71-3D-D330A were cotransfected into HeLa cells, respectively, with T7 RNA polymerase (plasmid pBABE). Cells were harvested 48 h after…

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  FIG 3. EV71 3CD upregulates the mRNA and protein of GADD34. (A) HeLa cells were transfected with pCE-puro-3×Flag-3CD and 3CD-Y441S. After 48 h, cells were harvested, and the mRNA level of GADD34 was detected by RT-qPCR. (B) The EV71 3CD was transfected into HeLa cells, and the cells were harvested 30 h after transfection to detect the level of GADD34 protein by Western blotting. Before being harvested, cells were treated with 10 μg/mL cycloheximide (CHX) for 0 h, 1 h, 2 h, and 4 h. (C) In the presence or absence of stress, the effect of uORF2 in the 5′-UTR region on the translation of the downstream main open reading frame (mORF). (D) GADD34 5′-UTR was constructed into a pGL3-SV40 promoter vector. On this basis, the AUG of two uORFs in the GADD34 5′-UTR region was mutated to AUA, respectively. (E and F) The plasmids constructed in (D) were transfected into HeLa cells, and the cells were harvested 30 h later. Before cell harvest, cells were treated with dimethyl sulfoxide (DMSO) or 10 mM dithiothreitol (DTT) for 6 h. Cell lysates were used to detect FL and β-galactosidase (β-gal) activities (E) and to detect proteins by Western blotting (F). (G and H) The plasmids constructed in (D) and…

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  FIG 4. GADD34 promotes EV71 replication, which was related to its PEST repeats area. (A) RD cells were transfected with siCon or siGADD34. 24 h after transfection, cells were infected with 1 MOI EV71 for 8 h. In harvested cells, proteins were detected by Western blotting. (B) The infectious clone pSVA-EV71, T7 RNA polymerase, and pCMV-3×HA vector or pCMV-3×HA-GADD34 were cotransfected into RD cells. Cells were harvested 48 h later, and the expression of EV71 VP1 was detected by Western blotting. (C) The HA vector or HA-GADD34 was transfected into RD cells. Cells were infected with 0.1 MOI EV71 24 h after transfection, and the culture supernatant was collected at 24 h, 48 h, and 60 h postinfection. The viral titers were measured by endpoint titration analysis and expressed as 50% tissue culture infectious dose (TCID50). Values were the means plus standard errors of the means (SEM) (error bars) from three individual experiments. Values were statistically evaluated using a two-way ANOVA. *, P < 0.05; **, P < 0.01. (D) Schematic diagram of GADD34 protein structure. The truncations are shown. (E to G) HA-GADD34 wild-type or various mutants, deletions, and truncations were transfected into RD…

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  FIG 5. GADD34 promotes EV71 IRES-mediated translation. (A) Schematic diagram of EV71 subgenomic replicon EV71-Luc. (B) The plasmids EV71-Luc or EV71-3D-D330A-Luc and HA vector or HA-GADD34 were cotransfected into HEK293T cells, then FL and β-gal activities were detected 48 h later. (C) HeLa cells were infected with pSIREN-RetroQ-shGADD34 or shControl (shCon) packaged lentivirus. Two days later, cells were transfected with pRHF-EV71 5′-UTR. Cells were collected 48 h after transfection to detect FL and RL activities, and Western blotting was used to detect the knockdown effect of GADD34. (D and E) HA-GADD34 wild-type or various mutants and pRHF-EV71 5′-UTR were transfected into HeLa cells. FL and RL activities were detected after 48 h. (F) HA-GADD34 wild-type or HA-GADD34 ΔPEST 1 and dual-luciferase plasmids containing several viral IRESs were cotransfected into HeLa cells. FL and RL activities were detected after 48 h. Values were the means plus standard errors of the means (SEM) (error bars) from two (F) or three (B, C, D, and E) individual experiments. Values were statistically evaluated using a two-way ANOVA (B, D, and E) or a two-tailed unpaired t test (C) or a one-way ANOVA (F). *,…

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  FIG 6. The C-terminal of GADD34 interacts with EV71 3CD. (A to C) 3×Flag-3CD (A), 3×Flag-3C-E71A (B), or 3×Flag-3D (C) and HA-GADD34, HA-GADD34-Δ563-565, or HA-GADD34-Δ555-567 were co-transfected into HEK293T cells. After 48 h, cells were lysed, the proteins were collected by co-immunoprecipitation and detected by Western blotting. (D) HA-GADD34 or HA-GADD34-Δ555-567 and 3×Flag-3CD were co-transfected into HeLa cells. After 48 h, cells were fixed, and the protein localization was detected by immunofluorescence. Bars represent 10 μm. (E) HA vector, HA-GADD34, HA-GADD34-Δ563-565, or HA-GADD34-Δ555-567 was transfected into RD cells. After 24 h, cells were infected with 1 MOI EV71 for 8 h, and the viral protein VP1 was detected by Western blotting.

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  FIG 7. EV71 3CD recruits GADD34 to EV71 5′-UTR. (A) HA-GADD34 or 3×Flag-hnRNP K and pRHF-EV71 5′-UTR were cotransfected into HEK293T cells. After 48 h, cells were lysed for RNA-protein immunoprecipitation (RIP). Agarose gel electrophoresis showed the amount of cDNA reverse-transcribed from the precipitated RNA. (B) HA vector, HA-GADD34, or HA-GADD34-Δ555-567 and pRHF-EV71 5′-UTR and 3×Flag-3CD were cotransfected into HEK293T cells. After 48 h, cells were lysed for RIP. Proteins were precipitated by anti-HA antibody and beads, and the cDNA reverse-transcribed from the pulled RNA was detected by agarose gel electrophoresis. (C) Summary chart of this study. EV71 upregulated the RNA and protein of host protein GADD34 through the non-structural precursor 3CD. 3CD recruited GADD34 to EV71 5′-UTR by interacting with GADD34 to promote IRES-mediated viral protein translation. GADD34 555 to 567 aa mediated the interaction with EV71 3CD, and PEST repeats contributed to the enhancement of IRES activity.