Foot-and-Mouth Disease Virus Capsid Protein VP1 Interacts with Host Ribosomal Protein SA To Maintain Activation of the MAPK Signal Pathway and Promote Virus Replication

Figures

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  FIG 1. FMDV VP1 interacted with host RPSA protein. (A and B) HEK-293T cells were cotransfected with 5 μg of Myc-vector-, Myc-VIM-, Myc-RPSA-, Myc-ESD-, or Myc-TPM4-expressing plasmids, and 5 μg of Flag-VP1-expressing plasmids for 36 h. The cells were then lysed and immunoprecipitated by anti-Myc antibody (A) or anti-Flag antibody (B). The immunoprecipitation (IP) complexes and whole-cell lysates (WCLs) were subjected to Western blotting using an anti-Myc, anti-Flag, or anti-β-actin antibody. (C) PK-15 cells were mock infected or infected with FMDV for 12 h, the subcellular localization of RPSA and FMDV VP1 was analyzed by immunofluorescence assay. Anti-VP1 (green) and anti-RPSA (red) antibodies and DAPI (blue) were used to stain the cells. (D and E) PK-15 cells were mock infected or infected with FMDV for 12 h. The cell lysates were then immunoprecipitated by anti-RPSA (D) or anti-VP1 (E) antibody. The IP complexes and WCLs were subjected to Western blotting with the anti-RPSA and anti-VP1 antibodies.

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  FIG 2. RPSA was not responsible for FMDV entry. (A and B) BHK-21 and PK-15 cells were incubated with 10 μg/ml of BSA or 1 or 10 μg/ml of the anti-N terminus (A) or anti-C terminus (B) of RPSA antibodies for 1 h and then infected with FMDV (MOI of 0.5) for 12 h. The FMDV RNA levels were measured by qPCR. (C) BHK-21 and PK-15 cells were collected and lysed, respectively. The expression of RPSA was detected by using the two anti-RPSA antibodies. (D) The cell membrane proteins were extracted from the BHK-21 and PK-15 cells. The membrane fractions were then immunoprecipitated with anti-RPSA antibody and subjected to Western blotting. (E) HEK-293T cells were transfected with 0, 0.5, 1, or 2 μg of Myc-RPSA-expressing plasmids for 24 h. The cells were then infected with FMDV at an MOI of 1 for 12 h. The cell lysates were subjected to Western blotting with anti-VP1, anti-Myc, and anti-β-actin antibodies. (F) HEK-293T cells were transfected with 2 μg of vector or Myc-RPSA-expressing plasmids for 36 h. The cell membrane proteins were immunoprecipitated with anti-Myc antibody and subjected to Western blotting.

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  FIG 3. RPSA performed an antiviral role during FMDV replication. (A and B) PK-15 cells were transfected with 0, 1, or 2 μg of Myc-RPSA-expressing plasmids for 24 h, followed by infection with FMDV at an MOI of 0.5 for 12 h. (A) The cells were subjected to Western blotting with anti-RPSA, anti-Myc, anti-VP1, and anti-β-actin antibodies. (B) FMDV RNA levels were detected using qPCR, and the viral yields were determined by using a 50% tissue culture infective dose (TCID50) assay. (C) PK-15 cells were mock transfected or transfected with 120 nM NC siRNA or RPSA siRNA (siRNA-1 or siRNA-2) for 36 h. The knockdown efficiency was then determined by qPCR and Western blotting. (D) PK-15 cells were transfected with NC siRNA, siRNA-1, or siRNA-2 for 36 h, followed by infection with FMDV at an MOI of 0.5 for 12 h. The mRNA expression levels of RPSA and FMDV were then measured by qPCR. The protein expression levels of RPSA and FMDV VP1 proteins were detected by Western blotting. The FMDV yields were determined by a TCID50 assay.

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  FIG 4. The activation of MAPK signaling pathway was essential for FMDV replication. (A and B) PK-15 cells were mock infected or infected with FMDV at an MOI of 0.5 for 0, 2, 4, 8, 12, or 16 h. The cells were then lysed and subjected to Western blotting. (A) The expression of RPSA and VP1 proteins was detected by using anti-RPSA and anti-VP1 antibodies, respectively. (B) The expression levels of MAPK pathway related proteins, including JNK1/2, ERK1/2, and p38 kinases and phosphorylated JNK1/2, ERK1/2, and p38 kinases (p-JNK1/2, p-ERK1/2, and p-p38), were detected by Western blotting. (C) PK-15 cells were pretreated with DMSO (solvent control) or 20 μM U0126 for 1 h and then infected with FMDV for 8 h. The expression levels of p-ERK1/2 and FMDV VP1 proteins were detected by Western blotting. (D and E) PK-15 cells were pretreated with DMSO or U0126 for 1 h and then infected with FMDV for 0, 4, or 8 h. (D) The expression levels of p-ERK1/2 and FMDV VP1 proteins were detected by Western blotting. (E) The mRNA expression levels of CCL2, CCL5, IL-6, TNF-α, and FMDV were measured by qPCR.

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  FIG 5. RPSA inhibited the activation of MAPK pathway during FMDV infection. (A) PK-15 cells were transfected with 2 μg of Myc-RPSA-expressing plasmids or empty vector for 24 h and then infected with FMDV for 0, 4, or 8 h. The expression levels of Myc-RPSA, JNK1/2, ERK1/2, and p38 kinases p-JNK1/2, p-ERK1/2, p-p38, and FMDV VP1 proteins were detected by Western blotting. (B) PK-15 cells were transfected with NC siRNA or RPSA siRNA (siRNA-2) for 36 h, followed by infection with FMDV for 0, 4, or 8 h. The expression levels of RPSA, JNK1/2, ERK1/2, and p38 kinases and p-JNK1/2, p-ERK1/2, p-p38, and FMDV VP1 proteins were detected by Western blotting. (C) PK-15 cells were transfected with NC siRNA or RPSA siRNA for 36 h and then treated with DMSO or U0126 for 1 h. The cells were infected by FMDV for 0, 4, or 8 h. The expression levels of RPSA, ERK1/2, p-ERK1/2, and FMDV VP1 proteins were detected by Western blotting. (D) PK-15 cells were transfected with 2 μg of Myc-RPSA-expressing plasmids or empty vector for 24 h and then infected with FMDV for 0, 4, or 8 h. The mRNA expression levels of CCL2, CCL5, IL-6, TNF-α, and FMDV were measured by qPCR. The FMDV yields at 8 hpi were determined by a…

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  FIG 6. FMDV VP1 interacted with RPSA to antagonize RPSA-mediated suppressive effect on MAPK signal pathway activation. (A) PK-15 cells were cotransfected with Myc vector or Myc-RPSA-expressing plasmids and Flag vector- or Flag-VP1-expressing plasmids, followed by infection with FMDV for 0, 4, or 8 h. The expression levels of Myc-RPSA, Flag-VP1, JNK1/2, ERK1/2, and p38 kinases and p-JNK1/2, p-ERK1/2, p-p38, and FMDV VP1 proteins were detected by Western blotting. The FMDV yields at 8 hpi were determined by a TCID50 assay. (B) Schematic presentation of a series of VP1 truncated mutants. (C) HEK-293T cells were cotransfected with 5 μg of Myc-RPSA and 5 μg of Flag vector, Flag-VP1, or Flag-tagged VP1 truncated mutants expressing plasmids for 36 h. The cells were then lysed and immunoprecipitated by anti-Flag antibody. The IP complexes and WCLs were subjected to Western blotting. (D) Schematic presentation of a series of VP1 truncated mutants. (E) HEK-293T cells were cotransfected with 5 μg of Myc-RPSA and 5 μg of Flag vector, Flag-VP1, or Flag-tagged VP1 truncated mutants expressing plasmids for 36 h. The cells were then lysed and immunoprecipitated with anti-Myc antibody. The IP complexes…