The Host Protein CAD Regulates the Replication of FMDV through the Function of Pyrimidines’ De Novo Synthesis

Figures

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  FIG 1. HDAC1 contributes to FMDV replication. (A) qRT-PCR was performed to detect the mRNA levels of viral 3D protein and host HDAC1 after FMDV infection. (B) Western blotting was performed to detect the protein levels of 3D and HDAC1 after FMDV infection. (C) qRT-PCR was performed to detect the mRNA levels of 3D and HDAC1 after plasmid-mediated overexpression of HDAC1. (D) Western blotting was performed to detect the protein levels of 3D and HDAC1 after overexpression of HDAC1. GAPDH was used as reference. n = 3. *, P < 0.05.

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  FIG 2. FMDV infection inhibited the HDAC1-AKT-mTOR axis. (A) Western blotting was performed to detect the protein levels of viral 3D protein, host HDAC1, and key proteins of the AKT-mTOR signaling pathway after FMDV infection. (B) Western blotting was performed to detect the protein levels of 3D, HDAC1, and key proteins of the AKT-mTOR signaling pathway after plasmid-mediated overexpression of HDAC1. (C) Cell counting kit-8 assay was performed to detect the cytotoxicity of different concentrations of bpV(HOpic) in BHK-21 cells. (D) Western blotting was performed to detect the protein levels of 3D, HDAC1, and key proteins of the AKT-mTOR signaling pathway after bpV(HOpic) treatment in BHK-21 cells. (E) Cell counting kit-8 assay was performed to detect the cytotoxicity of different concentrations of SC79 in BHK-21 cells. (F) Western blotting was performed to detect the protein levels of 3D, HDAC1, and key proteins of the AKT-mTOR signaling pathway after SC79 treatment in BHK-21 cells. GAPDH was used as a reference. n = 3.

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  FIG 3. CAD was induced by HDAC1 and regulated the replication of FMDV. (A) qRT-PCR was performed to detect the mRNA levels of host proteins downstream of the AKT-mTOR signaling pathway after FMDV infection. (B) qRT-PCR was performed to detect the mRNA levels of viral 3D protein and host CAD after FMDV infection. (C) Western blotting was performed to detect the protein levels of 3D and CAD after FMDV infection. (D) qRT-PCR was performed to detect the mRNA levels of 3D and CAD after shRNA-induced downregulation of CAD. (E) Western blotting was performed to detect the protein levels of 3D and CAD after downregulation of CAD. (F) TCID50 assay was performed to detect the titer of infectious virus. (G) Western blotting was performed to detect the protein level of CAD after bpV(HOpic) treatment in BHK-21 cells. (H) Western blotting was performed to detect the protein level of CAD after SC79 treatment in BHK-21 cells. (I) Western blotting was performed to detect the protein levels of 3D, CAD, and HDAC1 after plasmid-mediated overexpression of HDAC1. GAPDH was used as a reference. n = 3. *, P < 0.05.

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  FIG 4. CAD interacts with FMDV 2C protein in the replication complex. (A) Coimmunoprecipitation and Western blotting were performed to detect the interaction of CAD with FMDV proteinVP0, 3D, 2A+2B, 2C, VP1, VP3, 3A+3B, and 3C. (B) Coimmunoprecipitation and Western blotting were performed to detect the interaction of CAD and FMDV 2C protein. (C) Immunofluorescence was performed to detect the colocalization of CAD, 2C, and FMDV double-stranded RNA. n = 3.

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  FIG 5. The ACTase domain of CAD plays a critical role in FMDV replication. (A) Schematic design of a truncated mutant with a single domain of CAD removed. (B) Coimmunoprecipitation and Western blotting were performed to detect the interaction of FMDV 2C protein and truncated CAD mutants with a single domain removed. (C) Schematic design of a truncated mutant containing only a single domain of CAD. (D) Coimmunoprecipitation and Western blotting were performed to detect the interaction of FMDV 2C protein and truncated CAD mutants containing only a single domain. n = 3.

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  FIG 6. The regulation of viral replication by CAD is dependent on the pyrimidine synthesis function of CAD. (A) qRT-PCR was performed to detect the mRNA levels of CAD and viral 3D protein after shRNA-induced downregulation of CAD and uracil supplementation in BHK-21 cells. (B) Western blotting was performed to detect the protein levels of CAD and 3D after downregulation of CAD and uracil supplementation in BHK-21 cells. (C) TCID50 assay was performed to detect the titer of infectious virus. (D) Western blotting was performed to detect the protein level of 3D after plasmid-mediated overexpression of truncated CAD with a single domain removed and uracil supplementation in BHK-21 cells. (E) Western blotting was performed to detect the protein level of 3D after overexpression of a truncated CAD containing only a single domain and uracil supplementation in BHK-21 cells. GAPDH was used as a reference. n = 3. *, P < 0.05.

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  FIG 7. Schematic diagram depicting the regulation of FMDV replication by host CAD protein.