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The multiple functions of FMDV 3D during viral infection remain unclear. To better understand the role of FMDV 3D in viral replication, we aimed to identify host proteins that interact with 3D using the yeast two-hybrid system. Several host proteins were identified as potential targets of FMDV 3D (Table 1). One of these host proteins, identified as porcine host protein DDX1 (NCBI reference sequence GACC01000503.1) was selected for further study.
Gene Description Accession number DDX1 DEAD-box RNA helicase 1 GACC01000503.1 VDAC1 Voltage-dependent anion channel 1 AF268461.1 ATXN3 Ataxin 3 NM_001123081.1 Table 1. Porcine proteins identified as the potential target of FMDV 3D by the yeast-two hybrid.
To confirm the interaction between DDX1 and 3D, PK- 15 cells were transfected with FLAG-3D expressing plasmid or empty FLAG vector. The cell lysates were immunoprecipitated with anti-DDX1 antibody and analyzed by Western blotting. As shown in Fig. 1A, DDX1 pulled down FLAG-3D. A reverse immunoprecipitation experiment was also performed using anti-FLAG antibody. Similarly, FLAG-3D also immunoprecipitated with DDX1 (Fig. 1B).
Figure 1. DDX1 interacts with FMDV 3D protein. A PK-15 cells were seeded in 10-cm dishes, and the monolayer cells were transfected with 10 μg FLAG-3D expressing plasmid or 10 μg empty FLAG vector. The cells were lysed 32 h post-transfection (hpt) and cell lysates were immunoprecipitated with anti-DDX1 antibody. The whole-cell lysates and immunoprecipitated antibody-antigen complexes were analyzed by immunoblotting using anti-DDX1 and anti-FLAG antibodies. Ig represents IgG. B Similar infection and immunoprecipitation experiments were performed as described above. However, the lysates were immunoprecipitated with anti-FLAG antibody and analyzed by Western blotting. Ig represents IgG. C PK-15 cells cultured in 10-cm dishes were mock-infected or infected with FMDV (MOI = 0.5) for 12 h. The cell lysates were immunoprecipitated with antiDDX1 antibody. The antibody-antigen complexes were detected using anti-DDX1 or anti-3D antibodies. D A reverse immunoprecipitation was performed using anti-3D antibody as (C) described. The antibody-antigen complexes were detected by indicated antibodies. E HEK293T cells were cultured in Nunc glass bottom dishes, and the monolayer cells were transfected with 1.5 μg FLAG-3D expressing plasmid, 1.5 μg Myc-DDX1 expressing plasmid, or 1.5 μg empty vector. At 32 hpt, the expression of FLAG-3D and Myc-DDX1 was detected by IFA. Cells were double-immunostained for FLAG-3D (red) and Myc-DDX1 (green); cellular nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (blue).
To confirm the interaction between DDX1 and 3D in the context of viral infection, PK-15 cells were mock-infected or infected with FMDV at a multiplicity of infection (MOI) of 0.5. The cell lysates were immunoprecipitated with antiDDX1 antibody. DDX1 pulled down 3D in FMDV-infected cells (Fig. 1C). A reverse immunoprecipitation experiment was subsequently performed using anti-3D antibody, which showed that 3D also immunoprecipitated DDX1 (Fig. 1D). The results confirmed the 3D-DDX1 interaction in the context of viral infection.
The subcellular colocalization of 3D and DDX1 was also examined by IFA. The results indicated an interaction between 3D and DDX1 (Fig. 1E). Taken together, these results confirm that FMDV 3D interacts with DDX1.
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PK-15 cells were infected or mock-infected with FMDV (MOI = 0.5), and the DDX1 mRNA levels and protein abundance were compared at different time points. The expression of DDX1 mRNA, viral RNA, and viral titers gradually increased as infection progressed, whereas the abundance of the DDX1 protein was reduced over time (Fig. 2A). Meanwhile, a lower molecular weight band was observed as infection progressed (Fig. 2A).
Figure 2. FMDV infection reduces DDX1 protein expression. PK-15 cells were seeded in six-well plates and the monolayer cells were infected with FMDV (MOI = 0.5) (A) or mockinfected (B). The cells were collected at 0, 4, 8, 12, and 16 h. Expression of viral RNA and titers were determined by qPCR and TCID50, respectively (A); expression of DDX1 mRNA and protein were determined by qPCR and Western blotting, respectively (A, B). **P < 0.01 versus negative control.
Expression of DDX1 protein and mRNA remained unchanged in the mock-infected cells (Fig. 2B). Taken together, these results indicate that FMDV infection reduces DDX1 protein expression.
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To assess the roles of DDX1 on FMDV replication, PK-15 cells were transfected with different doses of Myc–DDX1 expressing plasmid. At 24 h post-transfection (hpt), cells were infected with FMDV (MOI = 0.5) for 12 h. As shown in Fig. 3A, the over-expression of DDX1 significantly suppressed FMDV replication in a dose-dependent manner.
Figure 3. DDX1 inhibits FMDV replication. A PK-15 cells were seeded in six-well plates and the monolayer cells were transfected with 1 or 3 μg Myc–DDX1 expressing plasmid or 3 μg empty Myc vector. At 24 hpt, the cells were infected with FMDV (MOI = 0.5) for 12 h. Expression of viral VP1 protein was determined by Western blotting and quantified as described; expression of viral RNA was determined by qPCR; viral titers were determined by TCID50. B, C PK-15 cells were seeded in six-well plates and the monolayer cells were transfected with 150 nmol/L nontargeting control (NC) siRNA or DDX1 siRNA for 36 h followed by infection with FMDV (MOI = 0.5) for 0, 6, and 12 h. Expression of viral RNA and DDX1 mRNA was determined by qPCR assay; the viral titers were determined by TCID50 (B). Expression of DDX1, IRF3, P-IRF3, and viral VP1 proteins were detected by Western blotting (C). **P < 0.01 versus negative control.
FMDV yields were further assessed. PK-15 cells were transfected with DDX1 or NC siRNA for 36 h, and then infected with FMDV (MOI = 0.5). The viral RNA, titers, VP1 protein, and the DDX1 protein were determined at 0, 6, and 12 h after FMDV infection. FMDV replication levels were significantly increased in the DDX1 siRNAtransfected cells compared with those in the NC siRNAtransfected cells (Fig. 3B, 3C). Taken together, these results demonstrate that DDX1 can inhibit FMDV replication.
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To analyze whether the ATPase/helicase activity of DDX1 was essential for the inhibition of FMDV replication, a Glu–Gly mutation (DDX1–DGAD) in the conserved DEAD-box motif was generated to abolish the ATPase/ helicase activity, as described previously (Tetsuka et al. 2004; Ishaq et al. 2009). PK-15 cells were transfected with Myc–DDX1 expressing plasmid, Myc–DDX1-DGAD mutant expressing plasmid, or empty vector, and then infected with FMDV (MOI = 0.5) for 0, 6 and 12 h. Viral RNA, titers, and protein levels were compared. As shown in Fig. 4, the DDX1–DGAD mutant exerted a slight inhibitory effect on FMDV replication, compared to that of wild-type DDX1. Taken together, these results indicate that DDX1-dependent inhibition of FMDV replication might require DDX1 ATPase/helicase activity.
Figure 4. Inhibition of FMDV replication partly depends on the ATPase/ helicase activity of DDX1. PK-15 cells were seeded in six-well plates and the monolayer cells were transfected with 2 μg Myc–DDX1 expressing plasmid, 2 μg Myc–DDX1-DGAD mutant expressing plasmid, or 2 μg empty Myc vector. At 24 hpt, the cells were infected with FMDV (MOI = 0.5) for 0, 6, and 12 h. Expression of viral VP1 and DDX1 protein was determined by Western blotting; expression of viral RNA was determined by qPCR; viral titers were determined by TCID50. *P < 0.05 and **P < 0.01 versus negative control.
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DDX1 can regulate innate immunity (Zhang et al. 2011; Fullam and Schroder 2013; Gu et al. 2017). Thus, we asked whether DDX1 was involved in FMDV-induced IFN-β activation. IFN regulatory factor (IRF)-3 phosphorylation is essential for regulating type Ⅰ IFN gene expression (Tian et al. 2018). Therefore, we examined the impact of DDX1 on the phosphorylation of IRF3 during FMDV infection. IRF3 phosphorylation levels were decreased in DDX1 siRNA-transfected cells, compared with that in NC siRNAtransfected cells (Fig. 3C).
The mRNA levels of IFN-β, OAS1, ISG54, and MX1 were also evaluated by qPCR. This revealed that the mRNA levels of IFN-β, OAS1, ISG54, and MX1 were reduced in DDX1 siRNA-transfected cells compared with those in NC siRNA-transfected cells (Fig. 5). Taken together, these results indicate that DDX1 is involved in FMDV-induced IFN-β production.
Figure 5. DDX1 is involved in FMDV-induced IFN-β activation. PK-15 cells were seeded in six-well plates and the monolayer cells were transfected with 150 nmol/L NC siRNA or DDX1 siRNA for 36 h followed by infection with equal amounts of FMDV (MOI = 0.5) for 0, 6, and 12 h. Expression of IFN-β, ISG54, OAS1, and MX1 mRNA was determined by qPCR assay. GAPDH was used as an internal control. *P < 0.05 and **P < 0.01 versus negative control.