Foot-and-mouth disease virus VP1 target the MAVS to inhibit type-I interferon signaling and VP1 E83K mutation results in virus attenuation

Fig 1. Wild-type FMDV VP1 negatively regulate type-I IFN pathway. PK15 cells were transiently transfected with wild-type VP1 and VP1 83K of FMDV O1 Manisa strain along with control vector, and VSV-GFP (1MOI) were infected. (A) GFP expression, (B) GFP absorbance and virus titer, and (C) IFN-β secretion was measured at indicated time points. The same experiment was conducted for the wild-type VP1 and VP1 83K of O/Andong/SKR/2010 (Andong) FMDV strain and similarly, (D) GFP expression, (E) GFP absorbance and virus titer, and (F) IFN-β secretion was measured. (G-H) Wild-type VP1 and VP1 83K of FMDV O1 Manisa and Andong strains along with control vector were transiently transfected to the PK15 cells, and VSV-GFP (1MOI) was infected. At 12 and 24hpi, cells were harvested and analyzed by quantitative real-time PCR analysis for IFN-β, IFN-α, IL-6, ISG-15, MCP1, IL-1β, PKR, and MX-1 genes. Data are representative of three independent experiments, each with similar results. Error bars indicate the mean ± SD of two biological replicates. Student’s t test; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Fig 2. Wild-type FMDV VP1 targets MAVS to inhibits IFN-β promoter activity. HEK293T cells were transiently transfected with wild-type VP1 and VP1 83K of FMDV O1 Manisa strain along with control vector, and VSV-GFP (1MOI) were infected. (A) GFP expression, (B) GFP absorbance and virus titer, and (C) IFN-β secretion was measured at indicated time points. The same experiment was conducted for the wild-type VP1 and VP1 83K of O/Andong/SKR/2010 (Andong) FMDV strain and similarly, (D) GFP expression, (E) GFP absorbance and virus titer, and (F) IFN-β secretion was measured. (G-J) HEK293T cells were transfected with the firefly luciferase reporter plasmid encoding the IFN-β promoter, plus TK-Renilla plasmid and an increasing dose of flag-tagged (G and I) wild-type VP1 and (H and J) VP1 83K plasmids of O1 Manisa and Andong strains, plus expression plasmids for RIG-I, 2CARD, MAVS, TRAF3, TBK1 and IKK-ε, for 24h. Results are expressed relative to those of Renilla luciferase alone (internal control). Data are representative of at least two independent experiments, each with similar results. All the values are expressed as mean ± SD of two biological replicates. Student’s t test; *p < 0.05; **p <…

Fig 3. Only wild-type FMDV VP1 compete with TRAF3 to bind with MAVS. (A) Full length and deletion mutants of MAVS was subjected to (B) immunoprecipitation with wild-type FMDV VP1 (83E) of O/Andong/SKR/2010 (Andong) strain. Lysates of HEK293T cells transfected with control vector (GST) or GST-MAVS constructs comprising amino acid 1–80, 1–180, 180–540, and full form of MAVS with Strep-tagged wild-type VP1 were subjected to GST-PD (pulldown), followed by immunoblotting with an anti-Strep antibody. Whole-cell lysate (WCL) was immunoblotted with anti-Strep and anti-GST antibodies. (C) HEK293T cells were cotransfected with the control vector (Strep), GST-MAVS, and Strep-tagged wild-type (83E) and mutated (83K) VP1 plasmids of FMDV Andong strain. Cell lysates were subjected to Strep pulldown (PD), followed by immunoblotting with an anti-GST antibody. Whole-cell lysate (WCL) was immunoblotted with anti-Strep and anti-GST antibodies. (D) HEK293T cells were transfected with Flag-tagged wild-type VP1 (83E) or VP1 83K plasmids of FMDV Andong strain together with V5-tagged MAVS plasmid, followed by confocal microscopy assay with anti-Flag (red) and anti-V5 (green) antibodies. Nuclei were stained…

Fig 4. Construction and characterization of the chimeric virus. (A and B) Schematic depiction of the chimeric viruses. The chimeric clones pOm-83E and pOm-AD-83K have replaced the 83rd amino acid with 83E or 83K through point mutation, respectively. (C) PK15 cells were infected with pOm-AD-83K and pOm-AD-83E (0.05MOI), together with mock infection. At 12hpi, cells were harvested and analyzed by quantitative real-time PCR analysis for IFN-β, IFN-α, IL-6, ISG-15, MCP1, and IL-1β genes. (D) The pOm-AD-83K and pOm-AD-83E chimeric virus were infected to BHK-21, LFBK, and IBRS-2 cells. At the indicated time points virus titer was determined by RNA extraction and quantitative real-time PCR analysis. (E) BHK-21, LFBK, and IBRS-2 cells were infected with each virus at 105.0~101.0 TCID50/0.1mL with 10-fold dilutions and stained with crystal violet at 72h after virus infection. The images show the wells inoculated with each virus at 103.0 TCID50/0.1mL. (F) For the comparison of pathogenesis in suckling mice (n = 10), the survival rate was determined after challenge with recombinant FMDVs intraperitoneally. In C, data are representative of two independent experiments, each with similar results and…

Fig 5. Pathogenesis of the chimeric viruses in pigs. The challenge experiment was carried out with six 90-day-old Yucatan pigs. (A) Three pigs were inoculated with pOm-AD-83K (#233, #235, #236). (B) Another three pigs were inoculated with pOm-AD-83E (#232, #234, #237). Each virus was inoculated on the footpad at a concentration of 105.0 TCID50/0.1mL. The left Y-axis of the graph shows the amount of virus in sera and swab as log10 values and the right Y-axis shows the clinical index as the maximum value of 16 points.