Foot-and-Mouth Disease Virus 3A Protein Causes Upregulation of Autophagy-Related Protein LRRC25 To Inhibit the G3BP1-Mediated RIG-Like Helicase-Signaling Pathway

Figures

• 

  FIG 1. G3BP1 and G3BP1E284A inhibit FMDV genomic copies and titer. (A) Effects of overexpression of G3BP1 and G3BP1E284A on FMDV replication in PK-15 cells. G3BP1 and G3BP1E284A overexpression in PK-15 cells were infected with FMDV (MOI, 1.0) for the indicated times. FMDV genome replication was evaluated by RT-PCR. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. (B) Effects of overexpression of G3BP1 and G3BP1E284A on FMDV titers in PK-15 cells. G3BP1 and G3BP1E284A overexpression in PK-15 cells were infected with FMDV (MOI, 1.0) for the indicated times. The virus titers in the supernatants collected were determined by immunofluorescence assay and expressed as TCID50/0.1 ml. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. (C) FMDV inhibits the expression of G3BP1 and G3BP1E284A proteins. G3BP1 and G3BP1E284A overexpression in PK-15 cells were infected with FMDV (MOI, 1.0) for the indicated times. G3BP1, G3BP1E284A, FMDV 3A, and FMDV VP3 proteins were detected by immunoblotting analysis with indicated antibodies. (D) Effects…

• 

  FIG 2. Porcine G3BP1 inhibits the RLH signaling pathway upstream of VISA. (A and B) Effects of overexpression of porcine G3BP1 on SeV-triggered IFN-β promoter (A) and ISRE (B) activation. HEK293T cells (1 × 105) were transfected with the IFN-β reporter or ISRE (0.1 μg) and the indicated expression (0.1 μg) plasmids. Twenty hours after transfection, cells were infected with SeV or left uninfected for 12 h before luciferase assays were performed. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. EV, empty vector. (C and D) Dose-dependent effects of porcine G3BP1 on SeV-triggered activation of IFN-β promoter and ISRE. The experiments were similarly performed as in A. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. (E and F) Effects of G3BP1 and its mutants on SeV-triggered IFN-β promoter and ISRE activation. The experiments were similarly performed as in A. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. (G) G3BP1 inhibits SeV-triggered induction…

• 

  FIG 3. (A) The G3BP1 protein interacts with the FMDV 3A protein. HEK293T cells were transfected with the indicated plasmids for 20 h before coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. (B and C) Endogenous G3BP1 is associated with FMDV 3A in PK-15 and PAM cells. PK-15 (B) or PAM cells (C) were infected with FMDV (MOI, 1.0) for the indicated times. Coimmunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. (D) Colocalization of FMDV 3A protein with G3BP1. HEK293T cells were cotransfected with Flag-3A and Myc-G3BP1. One day after transfection, the cells were fixed and subjected to indirect immunofluorescence to detect Flag-3A (green) and Myc-G3BP1 (red) with mouse anti-Myc and rabbit anti-Flag antibodies. The position of the nucleus is indicated by 4′,6-diamidino-2-phenylindole (DAPI; blue) staining in the merged image. (E) Colocalization of FMDV 3A protein with endogenous G3BP1. PK-15 cells were mock infected or infected with FMDV. The cells were fixed at 12 h postinfection and subjected to indirect immunofluorescence to detect FMDV 3A protein (green) and G3BP1 (red) with mouse anti-3A protein and rabbit anti-G3BP1…

• 

  FIG 4. FMDV 3A increases FMDV genomic copies and titer. (A) Effects of FMDV 3A overexpression in PK-15 cells on FMDV replication. FMDV 3A overexpression in PK-15 cells were infected with FMDV (MOI, 1.0) for the indicated times. FMDV genome replication was evaluated by RT-PCR. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. (B) Effects of FMDV 3A overexpression on FMDV titers in PK-15 cells. FMDV 3A overexpression in PK-15 cells were infected with FMDV (MOI, 1.0) for the indicated times. The virus titers in the supernatants collected were determined by immunofluorescence assay and expressed as TCID50/0.1 ml. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. (C) Effects of FMDV 3A overexpression on FMDV proteins in PK-15 cells. FMDV 3A overexpression in PK-15 cells were infected with FMDV (MOI, 1.0) for the indicated times. Flag-3A and FMDV VP3 proteins were detected by immunoblotting analysis with indicated antibodies.

• 

  FIG 5. FMDV 3A inhibits G3BP1-mediated RLH signaling pathway. (A and B) Effects of FMDV 3A and its mutants on G3BP1-mediated RLH signaling pathway during SeV infection. HEK293T cells (1 × 105) were transfected with the IFN-β reporter or ISRE (0.1 μg) and the indicated expression (0.1 μg) plasmids. Twenty hours after transfection, cells were infected with SeV or left uninfected for 12 h before luciferase assays were performed. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. EV, empty vector. (C, D, and E) Effects of FMDV and its mutants on poly(I·C)-induced signaling pathway in PK-15 cells. PK-15 cells were transfected with poly(I·C) (1 μg/ml) for 18 h. Cells were left uninfected or infected with FMDV or its mutants (MOI, 1.0) for 12 h, and RT-PCR experiments were performed. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. (F, G, and H) Effects of FMDV and its mutants on secretion of IFN-β induced by poly(I·C) in PAM cells. The experiments were similarly performed as in C. The experiment shown is a representative experiment…

• 

  FIG 6. FMDV 3A degrades G3BP1 by autophagy. (A) Dose-dependent effects of G3BP1 on the expression of RIG-I in HEK293T cells. HEK293T cells (2 × 105) were transfected with the Flag-RIG-I (0.5 μg) and Myc-G3BP1 plasmids (0 μg, 0.1 μg, or 0.2 μg) for 24 h. The cell lysates were analyzed by immunoblotting with anti-Flag, anti-β-actin, or anti-Myc antibodies. (B) Dose-dependent effects of G3BP1 on the expression of MDA5 in HEK293T cells. The experiments were similarly performed as in A. (C) Dose-dependent effects of G3BP1 on the expression of VISA in HEK293T cells. The experiments were similarly performed as in A. (D) Dose-dependent effects of FMDV 3A on the expression of G3BP1 and G3BP1E284A in HEK293T cells. The experiments were similarly performed as in A. G3BP1s, G3BP1 and G3BP1E284A. (E) Effects of G3BP1 with or without FMDA 3A on the expression of RIG-I in HEK293T cells. HEK293T cells were transfected with indicated plasmids for 24 h. The cell lysates were analyzed by immunoblotting with anti-Flag, anti-β-actin, or anti-Myc antibodies. (F) Effects of G3BP1 with or without FMDA 3A on the expression of MDA5 in HEK293T cells. The experiments were similarly performed as in E. (G) FMDV 3A…

• 

  FIG 7. FMDV 3A degrades G3BP1 by the autophagy-related protein LRRC25. (A) Dose-dependent effects of LRRC25 on the expression of RIG-I in HEK293T cells. HEK293T cells (2 × 105) were transfected with the Flag-RIG-I (0.5 μg) and Myc-LRRC25 plasmids (0 μg, 0.1 μg, or 0.2 μg) for 24 h. The cell lysates were analyzed by immunoblotting with anti-Flag, anti-β-actin, or anti-Myc antibodies. (B) Dose-dependent effects of LRRC25 on the expression of MDA5 in HEK293T cells. The experiments were similarly performed as in A. (C) Dose-dependent effects of FMDV 3A on the expression of LRRC25 in HEK293T cells. The experiments were similarly performed as in A. (D) Dose-dependent effects of LRRC25 on the expression of G3BP1 in HEK293T cells. The experiments were similarly performed as in A. (E) Interaction between FMDV 3A or 3D and LRRC25 in mammalian overexpression system. HEK293T cells were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. (F) Interaction between G3BP1 and LRRC25 in mammalian overexpression system. The experiments were similarly performed as in E. (G and H) Interaction between G3BP1, FMDV 3A…

• 

  FIG 8. LRRC25 increases FMDV genomic copies and titer. (A) Effects of LRRC25 overexpression on FMDV replication in PK-15 cells. LRRC25 overexpression in PK-15 cells were infected with FMDV (MOI, 1.0) for the indicated times. FMDV genome replication was evaluated by RT-PCR. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. (B) Effects of overexpression of LRRC25 on FMDV titers in PK-15 cells. LRRC25 overexpression in PK-15 cells were infected with FMDV (MOI, 1.0) for the indicated times. The virus titers in the supernatants collected were determined by immunofluorescence assay and expressed as TCID50/0.1 ml. The experiment shown is a representative experiment of three independent experiments with mean ± SD of three technical replicates. (C) Effects of overexpression of LRRC25 on FMDV proteins in PK-15 cells. LRRC25 overexpression in PK-15 cells were infected with FMDV (MOI, 1.0) for the indicated times. Myc-LRRC25, FMDV 3A, and FMDV VP3 proteins were detected by immunoblotting analysis with the indicated antibodies. (D) Effects of LRRC25-RNAi on FMDV replication in PK-15 cells. LRRC25 knockdown in PK-15…

• 

  FIG 9. Picornaviral 3A degrades G3BP1 by the autophagy pathway. (A) Effects of SVV 3A, EMCV 3A, and EV71 3A on the expression of G3BP1. HEK293T cells were transfected with HA-G3BP1 (0.5 μg) and SVV 3A, EMCV 3A, or EV71 3A (0 μg, 0.2 μg, or 0.4 μg) for 24 h. Immunoblotting analyses were performed with the indicated antibodies. (B) SVV 3A degraded G3BP1 by the autophagy pathway. HEK293T cells were transfected with the indicated plasmids for 16 h. Cells were treated with dimethyl sulfoxide (DMSO) or 3-MA for 6 h. Immunoblotting analysis was performed with the indicated antibodies. (C) EV71 3A degraded G3BP1 by the autophagy pathway. The experiments were similarly performed as in B. (D) EMCV 3A degraded G3BP1 by the autophagy pathway. The experiments were similarly performed as in B. (E) Dose-dependent effects of SVV 3A on the expression of LRRC25 in HEK293T cells. HEK293T cells (2 × 105) were transfected with the Myc-LRRC25 (0.5 μg) and Myc-SVV 3A plasmids (0 μg, 0.1 μg, or 0.2 μg) for 24 h. The cell lysates were analyzed by immunoblotting with anti-β-actin or anti-Myc antibodies. (F) Dose-dependent effects of EV71 3A on the expression of LRRC25 in HEK293T cells. The experiments were…

• 

  FIG 10. Working model of FMDV 3A regulation of the G3BP1-mediated RLH signaling pathway. Following FMDV infection, 3A, LRRC25, G3BP1, and RIG-I/MDA5 formed a complex. 3A inhibited the expression of G3BP1 by upregulating the expression of LRRC25, which resulted in a reduction in the expression of RIG-I and MDA5. Ultimately, this reduced the production of IFN-β.