Ribosomal Protein L13 Participates in Innate Immune Response Induced by Foot-and-Mouth Disease Virus

Figures

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  Figure 1. DDX3 cooperates with RPL13 to promote the IRES-dependent activity and replication of FMDV in PK-15 cells. (A) FMDV-infected PK-15 cell lysates at 5 hpi (MOI= 1) were subjected to an immunoprecipitation assay with an anti-RPL13 or anti-DDX3 antibody. After the cells were washed and dissociated, RNA was extracted from them and analyzed with RT–PCR using primers specific for the FMDV IRES or 3D/3′UTR, RPS16, or GAPDH. (B) PK-15 cells transfected with either the indicated siRNA or FLAG-tagged plasmid were transfected with the bicistronic construct psiCHECK–FMDV. At 24 h posttransfection, the RLuc and FLuc activities were measured. (C) PK-15 cells pretreated with the indicated amounts of RK-33 and ketorolac salt for 30 h were transfected with bicistronic plasmid psiCHECK–FMDV for 24 h in the presence of the indicated inhibitor. The cell lysates were then subjected to a luciferase reporter assay. (D) Control and RPL13-depleted PK-15 cells were transfected with bicistronic plasmid psiCHECK–FMDV in the presence of the indicated concentrations of RK-33 and ketorolac salt. siRNA-treated cells were transfected with the empty FLAG vector or increasing amounts of plasmid encoding FLAG–DDX3…

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  Figure 2. Effects of RPL13 overexpression on FMDV replication. PK-15 (A) and BHK-21 cells (B) were transfected with plasmid encoding FLAG–EV or FLAG–RPL13 for 24 h, and then challenged with FMDV (MOI = 0.5). At various time points, the viral proteins were detected with western blotting, the viral RNA levels were determined with RT–qPCR, and the viral titers were determined with a TCID50 assay. The data reflect the mean SD of three separate trials (*p < 0.05, **p < 0.01).

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  Figure 3. RPL13 stimulates the innate antiviral response in DDX3-independent manner. (A–D) PK-15 cells were cotransfected with plasmid encoding FLAG–EV or FLAG–RPL13 and pGL4–IFN-β–Luc or pGL4–NF-κB–Luc, together with the pRL-TK plasmid as the internal control. Cells treated with the control siRNA (siCtrl) or RPL13-targeting siRNA (siRPL13) for 48 h were cotransfected with a reporter plasmid and pRL-TK. At 24 h posttransfection, the cells were treated with poly(I:C) (1 μg/well) (A, B) or FMDV (MOI = 0.5) (C, D) for the indicated times. Dual luciferase activities in the cell lysates were then measured. (E, F) Control and DDX3-depleted PK-15 cells were cotransfected with plasmid encoding FLAG–EV or FLAG–RPL13 and pGL4–IFN-β–Luc (E) or pGL4–NF-κB–Luc (F), together with pRL-TK plasmid. At 24 h posttransfection, the cells were treated with poly(I:C) (1 μg/well) for the indicated times. The cell lysates were then subjected to a dual luciferase reporter assay. The data reflect the mean SD of three separate trials (*p < 0.05, **p < 0.01).

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  Figure 4. RPL13 promotes the expression of IFN-β and IL-6. PK-15 cells in which RPL13 was overexpressed or knocked down were infected with FMDV (MOI = 0.5). At the indicated times, total RNA was extracted to detect the levels of IFN-β, IL-6 and PKR mRNAs with RT–qPCR (A, B, E). The cell supernatants were collected to measure IFN-β and IL-6 protein secretion with ELISAs (C, D). The viral proteins were detected with western blotting (F). The data reflect the mean SD of three separate trials (*p < 0.05, **p < 0.01).

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  Figure 5. RPL13 is a specific target of FMDV proteinase 3Cpro. (A, B) PK-15 cells were infected with FMDV (MOI = 0.5). At the indicated times, total RNA was extracted and subjected to qPCR analysis (A), and the cell lysates were analyzed with western blotting (B). (C, D) PK-15 cells grown in a 10 cm dish were cotransfected with plasmid encoding GFP–RPL13 (7 μg) and one of various plasmids expressing FLAG-tagged viral proteins (Lpro, 2B, 2C, 3A, 3B, 3Cpro, or 3Dpol) or FLAG–EV (7 μg). At 30 h posttransfection, the cell lysates were immunoprecipitated with control IgG, anti-FLAG antibody (C), or anti-GFP (D) antibody. The cell lysates (input) and the precipitates were then subjected to western blotting with the indicated antibodies. (E) PK-15 cells were cotransfected with 2 μg of plasmid encoding FLAG–RPL13 and 2 μg of plasmid encoding HA–EV, HA–3Cpro, or various mutants of HA–3Cpro for 30 h. The cell lysates were subjected to western blotting with anti-FLAG, anti-HA, anti-G3BP1, or anti-β-actin antibody. (F) PK-15 cells were cotransfected with plasmid encoding FLAG–EV or FLAG–RPL13 and plasmid encoding HA–EV or HA–3Cpro in the presence or absence of MG132 (2 or 20 μM), CQ (50 or 100 μM), or…