Foot-and-Mouth Disease Virus Evades Innate Immune Response by 3C-Targeting of MDA5

Figures

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  Figure 1. Down-regulation of MDA5 protein expression by foot-and-mouth disease virus (FMDV) infection. The PK-15 cells (1.5 × 106 cells) were infected with FMDV (0.5 MOI) and viral RNA, total RNA and protein were extracted at different times (0, 4, 8, 12 and 24 hpi) after infection. (a) The levels of viral RNA were examined by real-time RT-PCR (left panel) and viral titers were determined by TCID50 assay (right panel). (b) The levels of MDA5 (left panel), RIG-I and IFN-β (right panel) mRNA in the cell lysates were determined by real-time RT-PCR normalized to GAPDH. (c) The levels of endogenous MDA5, RIG-I proteins and VP1 structural protein of FMDV were detected by Western blotting. (d) The expression levels of the IRF3 and p-IRF3 proteins were analyzed by Western blotting (left panel). All experiments were performed in triplicate and presented as mean ± SEM (*, p < 0.05; **, p < 0.01; ***, p < 0.001 and n.s., not significant). The abundance of proteins were calculated by Image J software to quantify the band intensity, normalized to β-actin and respectively compared with the 0 hpi.

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  Figure 2. The effects of Lbpro and 3Cpro on endogenous MDA5 protein expression. (a) HEK293T cells (1 × 106 cells) were transfected with HA (Hemagglutinin)-tagged proteins plasmids (MDA5, Lb, 2B, 2C, 3A, 3B, 3C or 3D) or empty HA-vector plasmid at the amount of 2 μg. Cells were lysed 16 h later and analyzed by Western blotting with anti-HA, MDA5, RIG-I and β-actin antibodies. (b) The abundance of MDA5 and RIG-1 proteins were calculated by Image J software to quantify the band intensity, normalized to β-actin and respectively compared with the group of HA-vector. The result is shown of one of the triplicate experiments, presented as mean ± SEM (*, p < 0.05 and n.s., not significant).

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  Figure 3. The effects of Lbpro and 3Cpro on exogenous MDA5 protein expression. (a) HEK293T cells (1 × 106 cells) were co-transfected with HA-tagged MDA5-expressing plasmid (2 μg) and various plasmids expressing HA-tagged viral nonstructural proteins (Lb, 2B, 2C, 3A, 3B, 3C or 3D) or empty HA-vector plasmid (2 μg). The expression of HA-tagged NSP and MDA5 was determined by Western blotting at 16 h. (b) HEK293T cells (1 × 106 cells) were transfected with HA-tagged MDA5-expressing plasmid (2 μg) along with different amounts of HA-Lb or 3C expressing plasmid (0, 0.5, 1 or 2 μg). The expression of MDA5 mRNA was detected by real-time RT-PCR normalized to GAPDH (upper panel) and levels of proteins were analyzed by Western blotting with anti-HA, MDA5 and β-actin antibodies at 16 h. The values of mRNA levels are presented as mean ± SEM (**, p < 0.01; ***, p < 0.001). (c) HEK293T cells (1 × 106 cells) were transfected with HA-tagged MDA5-expressing plasmid (2 μg) along with different dose of HA-NSP (2B, 2C or 3A) expressing plasmid (0, 0.5, 1 or 2 μg) for 16 h. The expression of HA-tagged NSP and MDA5 proteins in cell lysates were analyzed by Western blotting with anti-HA, MDA5 antibodies. All the…

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  Figure 4. The effects of proteasome, lysosome and caspase inhibitors on Lbpro- or 3Cpro-induced MDA5 reduction. (a) HEK293T cells (1.5 × 106 cells) were mock-transfected or were co-transfected with HA-MDA5-expressing plasmid (2 μg) and empty vector (2 μg) or HA-Lbpro expressing plasmids (2 μg). Cells were maintained for 12 h in the presence or absence of the proteasome inhibitor MG132 (2 or 20 μM), the caspase inhibitor Z-VAD-FMK (10 or 50 μM) or lysosome inhibitor chloroquine (CQ; 50 or 100 μM). (b) HEK293T cells (1.5 × 106 cells) were mock or co-transfected with HA-MDA5-expressing plasmid (2 μg) and empty vector (2 μg) or HA-3Cpro expressing plasmid (2 μg) and maintained in the presence or absence of MG132 (2 or 20 μM), Z-VAD-FMK (10 or 50 μM) or CQ (50 or 100 μM). The protein levels of the above-described experiments were determined by Western blotting with anti-HA, MDA5 and β-actin antibodies. All the data were repeated in three independent experiments and data from one of the triplicate experiments are shown.

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  Figure 5. The effects of catalytic residues H46, D84 and C163 of the 3Cpro active site on FMDV 3Cpro-induced MDA5 protein reduction. (a) HEK293T cells (1.5 × 106 cells) were transfected with HA-tagged empty vector, 3Cpro or individual mutants of 3Cpro (2 μg) for 12 h. (b) HEK293T cells (1.5 × 106 cells) were co-transfected with HA-tagged, MDA5-expressing plasmid (2 μg) and various plasmids expressing 3Cpro or individual mutants of 3Cpro (2 μg) for 12 h. The abundance of MDA5 proteins was calculated by Image J software to quantify the band intensity, normalized to β-actin and respectively compared with the group of HA-Vec. or HA-3Cpro. The result from one of the triplicate experiments are shown and presented as mean ± SEM (**, p < 0.01; ***, p < 0.001 and n.s., not significant).

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  Figure 6. Interaction between 3Cpro and MDA5. HEK293T cells grown in 10-cm dishes were co-transfected with FLAG-MDA5 plasmid (8 μL) and the indicated plasmids (8 μL). The cells were lysed at 10 h and immunoprecipitated with mouse anti-FLAG antibody or mouse normal IgG antibody and then subjected to western blotting with anti-FLAG or anti-HA antibodies. Whole cell lysates (WCL) were analyzed by immunoblotting (IB) using anti-FLAG or anti-HA antibodies. All the data were repeated in three independent experiments and data from one of the triplicate experiments are shown.