Inhibition of MAVS Aggregation-Mediated Type-I Interferon Signaling by Foot-and-Mouth Disease Virus VP3

Figure 1. FMDV VP3 negatively regulates the antiviral responses. (A) GFP expression, (B) GFP fluorescence and virus titer, and (C) IL-6 and IFN-β secretion were measured in FMDV VP3 stably expressing or control Raw264.7 cells after VSV-GFP (1MOI) infection. Similarly, (D,G) GFP expression, (E,H) GFP fluorescence and virus titer, and (F,I) IL-6 and IFN-β secretion were measured in control plasmid or FMDV VP3 plasmid transiently transfected HEK293T and PK15 cells after VSVS-GFP (1MOI) infection. All the values are given as mean ± SD of two biological replicates. Scale bar represents 50 μM. Data are representative of three independent experiments, each with similar results. Student’s t-test; * p < 0.05; ** p < 0.01; ns, not significant.

Figure 2. FMDV VP3 inhibits the activation of key molecules in the type-I IFN signaling cascade and suppresses antiviral gene expression. (A) Control plasmid or FMDV VP3 plasmid transiently transfected PK15 cells were infected with PR8-GFP (3MOI). At the indicated times after infection, TBK1, IRF3, STAT1, P65, and phosphorylated TBK1, IRF3, STAT1, and P65 protein levels in PK15 cell extracts were detected by immunoblotting, followed by the qRT-PCR for FMDV VP3, PR8-GFP virus NS1 protein, and β-actin. The β-actin was used to confirm the equal loading of proteins for immunoblotting. (B) HEK293T cells were transfected with control or FMDV VP3 plasmid together with GFP-IRF3 (green) plasmid, followed by infection with H1N1 influenza virus (1MOI). Cells were fixed at indicated time points and DAPI (blue) was used to stain the nuclei. Scale bar represents 5 μM. (C) The percentages of cells showing nuclear translocation of IRF3 were calculated by dividing the number of cells exhibiting nuclear expression of IRF3 by the total number of GFP-positive cells. (D) Control plasmid or FMDV VP3 plasmid transiently transfected PK15 cells were infected with PR8-GFP (3MOI). At indicated time points after…

Figure 3. FMDV VP3 binds to the transmembrane domain of MAVS. (A) HEK293T cells were transfected with an IFN-β reporter plasmid, plus TK-Renilla plasmid and increasing amounts of FMDV VP3 plasmid, along with overexpression plasmids for RIG-I, MDA5, MAVS, TRIF, TRAF3, or TBK1. At 24 h post-transfection, luciferase activity was measured in a luminometer. TK-Renilla was used as transfection control to normalize firefly luciferase activity. (B) HEK293T cells were cotransfected with the control vector (Flag), Flag-tagged RIG-I, MDA5, MAVS, and TBK1 plasmids together with GST-tagged FMDV VP3 plasmids. Cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting with an anti-GST antibody. WCL was immunoblotted with anti-Flag and anti-GST antibodies. (C) HEK293T cells were cotransfected with the control vector (GST), Flag-MAVS, and GST-tagged FMDV VP3 plasmid. Cell lysates were subjected to GST pulldown (PD), followed by immunoblotting with an anti-Flag antibody. Whole-cell lysate (WCL) was immunoblotted with anti-GST and anti-Flag antibodies. Lysates of (D) HEK293T cells transfected with control vector (Flag) or with Flag-tagged FMDV VP3 plasmid were subjected to…

Figure 4. FMDV VP3 disrupts MAVS activation. (A,B) Immunoblot analysis of mitochondria fraction isolated from the (A) PK15 and (B) HeLa cells which transfected with increasing amounts of V5-tagged FMDV VP3 expression plasmid, and Sendai virus (SeV) infected (1MOI). Mitochondria fraction was immunoblotted with the MAVS, Tom40, and α-tubulin antibodies. PK15 cell whole-cell lysate (WCL) was immunoblotted with MAVS and α-tubulin antibodies, while HeLa cell WCL was immunoblotted with anti-MAVS, anti-V5, and anti-α-tubulin antibodies. In PK15 cells, qRT-PCR was done to detect the expression of FMDV VP3. (C) HEK293T cells were cotransfected with control vector (Strep), Flag-MAVS, Strep-MAVS, and increasing doses of V5-tagged FMDV VP3 plasmid. The cell lysates were subjected to Strep-PD and subsequent immunoblotting with anti-Flag, anti-V5, and anti-Strep antibodies. Further, WCL was immunoblotted with anti-Flag, anti-Strep, and anti-V5 antibodies. (D) PK15 and (E) HeLa cells were transfected with increasing amounts of V5-tagged FMDV VP3 plasmid and infect the SeV (1MOI). Following that, crude mitochondria were isolated from the cells and subjected to the semi-denaturing detergent agarose gel…