Inhibition of SR Protein Phosphorylation Impairs IRES-Dependent Translation and Attenuates EV-A71 Replication.
<p>(A) Effects of SR protein kinase inhibitor (SRPKIN-1, 200 nM) and Clk kinase inhibitor (TG003, 50 μM) on SR protein phosphorylation and viral protein expression were analyzed by western blotting using pan-SR (α-SR) and pan-phospho-SR (α-pSR) antibodies. Unless otherwise indicated, the same...
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2025
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| Summary: | <p>(A) Effects of SR protein kinase inhibitor (SRPKIN-1, 200 nM) and Clk kinase inhibitor (TG003, 50 μM) on SR protein phosphorylation and viral protein expression were analyzed by western blotting using pan-SR (α-SR) and pan-phospho-SR (α-pSR) antibodies. Unless otherwise indicated, the same concentrations were used in all experiments shown. (B) Quantitative analysis of total and phosphorylated SR protein levels following drug treatments. GAPDH was used as a loading control. Data represent the mean ± SD from three independent experiments. Protein levels in the DMSO group were set as 100%. (C) Viral proteins were detected using α-3D<sup>pol</sup> and α-VP0/VP2 antibodies, with GAPDH as the loading control. (D) Quantification of viral VP0 and 3CD protein levels after drug treatment. Data are presented as the mean ± SD from three independent experiments. (E) Viral titers from EV-A71-infected RD cells (MOI 20, 12 hpi) treated with various drugs were determined by plaque assay. Results are shown as the mean ± SD from three independent experiments. (F) Schematic representation of the time-of-addition assay (upper panel). TG003 or SRPKIN-1 was added either before or after infection, as indicated. Viral titers were measured at 8 hpi and are shown as the mean ± SD from three independent experiments (lower panel). (G) Dual luciferase reporter assay using the pRHF-EV-A71 plasmid, which contains the EV-A71 IRES element positioned between the upstream Renilla luciferase (Rluc) and downstream Firefly luciferase (Fluc) coding sequences (schematic, upper panel). (H) RD cells were transfected with the reporter plasmid for 5 h, followed by EV-A71 infection (MOI 20, 4 h) under the indicated treatments. Relative Fluc/Rluc ratios in (G) and (H) are shown as the mean ± SD from a representative triplicate of at least three independent experiments. (I) Mono-luciferase assay using in vitro synthesized RNA containing the EV-A71 IRES upstream of the Fluc CDS (schematic, upper panel). Transfection of in vitro synthesized RNA was performed for 5 h, followed by EV-A71 infection at an MOI of 10 for 4 h under the indicated treatments. The Fluc values are shown as the mean ± SD from a representative triplicate of at least three independent experiments. (J) Plaque reduction assay evaluating the effect of SRPKIN-1/TG003 on other enteroviruses, including CV-A16, CV-B3, and EV-D68. Data are shown as the mean ± SD from three independent experiments. Statistical analysis was performed using the Student’s t-test. ****: p < 0.0001; ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: not significant.</p> |
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