Fig 1: EV71 infection stimulates EV71 IRES-dependent translation and DDX3X facilitates the translation. A schematic of 5'UTR-LucPEST-3'UTR monocistronic reporter RNA (A), or Cap-LucPEST reporter RNA (C), is shown on the top. The RD cells, treated with shCtrl or shDDX3X#003 (A,C), or with shCtrl, shDDX#003 or shDDX3X#004 (B), were mock-infected or infected with EV71 at an MOI of 5. After viral infection, the cells were further transfected with the in vitro transcribed 5'UTR-LucPEST-3'UTR reporter RNA (A,B) or Cap-LucPEST reporter RNA (C). The cells were harvested at the indicated time points post-EV71 infection (A,C) or at 7 h.p.i. (B). Half of the cells were used for luciferase activity assays, 1/10 for Alamar Blue assay to determine cell viability, and the rest were used for RNA quantification by RT-qPCR to determine the RNA levels. The luciferase activity was normalized against Alamar Blue value and the RNA levels. All the results presented are the mean ± SD of three independent experiments. One-way ANOVA followed by Bonferroni’s correction was performed. In (A), the statistical analyses indicated ***P <0.001 for EV71/shCtrl vs. Mock/shCtrl at all time points except 3 h.p.i., ***P <0.001 for EV71/shCtrl vs. EV71/shDDX3X at 7 h.p.i., and ***P <0.001 for Mock/shCtrl vs. Mock/shDDX3X at 9 h.p.i.
Fig 2: DDX3X is a positive regulator of EV71 replication. RD cells were first infected with lentiviruses expressing control shRNA (shCtrl) or shRNAs targeting DDX3X (shDDX3X#003 and shDDX3X#004). After 2 days of selection with puromycin, the cells were infected with EV71 (MOI = 5). (A) The knockdown efficiency of shDDX3X#003 and shDDX3X#004 analyzed by western blot analysis is shown. (B) EV71 viral titers were measured by plaque assays from the supernatant and cell lysates of EV71-infected cells at the indicated time points post-viral infection. (C) Viral RNA levels in EV71-infected cells were measured by RT-qPCR at 6 h.p.i. (D) Expression of the viral proteins in EV71-infected cells was analyzed at 6 h.p.i. using mouse antibodies against 2C, 3C, 3D, and VP1. The relative expression levels are indicated by the numbers. The western blot analysis of ß-actin, used as a loading control, was shared in (A,D). In (B,C), one-way ANOVA followed by Dunnett’s post hoc analysis was performed for comparison between the shCtrl group and the DDX3X knockdown groups. *P <0.05, **P <0.01, ***P <0.001.
Fig 3: DDX3X unwinds domain VI of EV71 IRES to facilitate IRES-dependent translation. (A) The secondary structure of domain VI of EV71 IRES is shown. The conserved Yn-Xm-AUG592 motif is highlighted in the boxes. (B) The secondary structures of domain VI for the wild-type (WT) and mutants M1–M4 predicted by M-fold software are shown. The mutated sequences are shown in capital letters. (C) DDX3X dependences of various IRES mutants were determined. The RD cells depleted of DDX3X (shDDX3X) or not (shCtrl) were infected with EV71 (MOI = 5); the cells were then transfected with the in vitro transcribed WT or mutant IRES-Luc reporter RNAs and the luciferase activity was measured at 6 h post EV71 infection. The reduction percentage of the luciferase activity by DDX3X knockdown for each mutant IRES is shown at the bottom. The results expressed are the mean ± SD of three independent experiments. Two-tailed Student’s t-test was performed for the comparison between the shCtrl and the shDDX3X groups within each IRES mutant, whereas one-way ANOVA followed by Dunnett’s post hoc test was performed between the indicated groups. *P <0.05, **P <0.01, ***P <0.001.
Fig 4: The DDX3X is required for breast cancer cells proliferation. (A) Western blot showing the protein levels of DDX3X and GAPDH in MCF7 cells transfected with either scrambled siRNA or one of two different siRNAs targeting DDX3X (#6 or #8). Cells were harvested at the indicated time points. Re-expression of DDX3X at day 4 is presumably due to loss of the transfected siRNAs. (B) Proliferation curves for MCF7 cells treated as in A. Equal numbers of cells (3 × 104) were seeded for proliferation assays 24 h after transfection (day 0) and then counted at the indicated time points. (C) Clonogenic assay of MCF7 cells treated as in A: 24 h after transfection, cells were seeded for clonogenic growth and stained 6 days after seeding. Images from a representative experiment are shown. (D) Quantification of number of colonies from C. P-values represent statistical significance calculated with unpaired t test comparing to scrambled siRNA condition (P-values: ns. >0.05; *=0.05; **=0.01; ***=0.001).
Fig 5: DDX3X stimulates the translation of other types of viral IRESs. The IRESs that direct the translation of luciferase reporter in the monocistronic constructs were derived from (A) CA16 virus, (B) Echovirus 9, (C) EMCV, and (D) HCV. The former three IRES-Luc RNAs were transfected into HeLa cells, whereas HCV IRES-Luc RNA was transfected into Huh7 cells. Both HeLa and Huh7 cells were pre-treated with lentiviruses expressing shCtrl or shDDX3X. The luciferase activity was measured at 6 h post RNA transfection. Results presented are the mean ± SD of three independent experiments. *P <0.05, ***P <0.001 between the shCtrl and the shDDX3X groups (Student’s t-test).
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