Foot-and-Mouth Disease Virus 3C Protease Antagonizes Interferon Signaling and C142T Substitution Attenuates the FMD Virus

FIGURE 1. Wild-type FMDV 3Cpro negatively regulates type-I IFN pathway. Wild-type FMDV 3Cpro stably expressing or control Raw264.7 cells were infected with VSV-GFP (1 MOI) at indicated time points and, (A) GFP expression, (B) FMDV 3Cpro expression, (C) GFP absorbance and virus titer, and (D) IL-6, IFN-β, IFN-α, and TNF-α secretion was measured. The same cell line was treated with (E) poly (I:C) or (F) 5′PPP-dsRNA and measured their IL-6 and IFN-β secretion. HEK293T cells were transiently transfected with control plasmid or wild-type FMDV 3Cpro plasmid, and VSV-GFP (0.5 MOI) were infected. At indicated time points (G) GFP expression, (H) FMDV 3Cpro expression, (I) GFP absorbance and virus titer, and (J) IL-6, IFN-β secretion was measured at indicated time points. Data are representative of three independent experiments, each with similar results. FMDV, foot-and-mouth disease virus; IFN, interferon; IL-6, interleukin 6; TNF-α, tumor necrosis factor-alpha. All the values are expressed as mean ± SD of three biological replicates. Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

FIGURE 2. Wild-type FMDV 3Cpro degrades RIG-I and MDA5. PK15 cells were transiently transfected with control plasmid or wild-type FMDV 3Cpro in the absence or presence of (A) MG132 (5, 10, 20 μM), (B) Z-VAD (10, 50, 100 μM), (C) CQ (50, 100, 200 μM), (D) NH4Cl (10, 20, 50 mM), and (E) Rupintrivir (10, 20, 40 μM). Cell lysate was subjected to immunoblotting with antibody against RIG-I and β-actin, following the qRT-PCR for FMDV 3Cpro and β-actin. (F) HEK293T cells were cotransfected with the Flag-tagged porcine RIG-I plasmid together with control plasmid or Strep-tagged FMDV 3Cpro plasmid in the absence or presence of Rupintrivir. The cell lysate was subjected to Strep pulldown followed by immunoblotting with individual antibodies against Flag, Strep, or β-actin. (G–K) PK15 cells were transfected with control plasmid or wild-type FMDV 3Cpro following the EV71 infection in the absence or presence of (G) MG132 (5, 10, 20 μM), (H) Z-VAD (10, 50, 100 μM), (I) CQ (50, 100, 200 μM), (J) NH4Cl (10, 20, 50 mM), and (K) Rupintrivir (10, 20, 40 μM) as indicated above. Cell lysates were subjected to immunoblotting against individual antibodies of MDA5 and β-actin, following the qRT-PCR for FMDV 3Cpro…

FIGURE 3. Wild-type FMDV 3Cpro mediated RIG-I and MDA5 degradation is related to its protease activity. HEK293T cells were transiently transfected with (A) Flag-tagged RIG-I or (B) MDA5 plasmid together with Strep-tagged control plasmid or Strep-tagged wild-type FMDV 3Cpro. At 24 h after transfection of plasmids, cells were treated with Cycloheximide (CHX) and cells were harvested at indicated time points after CHX treatment. Cell lysates were subjected to immunoblotting with individual antibodies against Flag, Strep, and β-actin. For the wild-type FMDV 3Cpro mediated in vitro RIG-I degradation, RIG-I protein (2 μg) was incubated with (C) control or (D) with FMDV-3C wild-type protein (2 μg) in the in vitro degradation buffer containing 50 mM HEPES-KOH pH 7.5, 35 mM KCl and 1 mM DTT. Reactions were carried out at 37°C for 0–2 h and terminated by adding an equal volume of SDS-PAGE sample buffer and heating at 100°C for 5 min. The samples were subjected to immunoblotting with anti-Flag, -RIG-I, and -Strep antibodies.

FIGURE 4. FMDV 3Cpro C142T is deficient in the negative regulation of type-I IFN signaling. The PK15 cells or LFBK cells were transiently transfected with wild-type FMDV 3Cpro, 3Cpro C142T, or control plasmid, following VSV-GFP (1 MOI) infection. (A,E) GFP expression, (B,F) wild-type FMDV 3Cpro and 3Cpro C142T expression by qRT-PCR, (C,G) GFP absorbance and virus titer, (D,H) IL-6 and IFN-α secretion was measured at indicated time points. (I) PK15 cells were transiently transfected with wild-type FMDV 3Cpro, 3Cpro C142T, or control plasmid and infected with PR8-GFP (3 MOI). Immunoblotting was performed with cells harvested at 0, 6, 12, and 18 hpi to detect phosphorylated (p-) TBK1, TBK1, p-IRF3, IRF3, p-STAT1, STAT1, p-IκB-α, IkB-α, and β-actin. (J) PK15 cells were transiently transfected with wild-type FMDV 3Cpro, 3Cpro C142T, or control plasmid and PR8-GFP (3 MOI) infection, following total RNA extraction and qRT-PCR for respective antiviral genes as mentioned. Data are representative of three independent experiments each with similar results. FMDV, foot-and-mouth disease virus; IFN, interferon; IL-6, interleukin 6; TBK1, TANK binding kinase 1; IRF3, interferon regulatory factor 3;…

FIGURE 5. FMDV 3Cpro C142T is not involved in RIG-I and MDA5 degradation. (A) HEK293T cells were transiently transfected with Strep-tagged wild-type FMDV 3Cpro or 3Cpro C142T plasmids in a dose-dependent manner, or control plasmid, together with Flag-tagged porcine RIG-I plasmid. Cell lysates were subjected to immunoblotting with individual antibodies against Flag, Strep, and β-actin, and (B) band intensity was measured. (C) PK15 cells were transiently transfected with Strep-tagged wild-type FMDV 3Cpro or 3Cpro C142T plasmids in a dose-dependent manner, or control plasmid. Cell lysates were subjected to immunoblotting with individual antibody against RIG-I, or β-actin, following the qRT-PCR for FMDV 3Cpro, 3Cpro C142T, and β-actin, and (D) band intensity was measured. (E) HEK293T cells were transfected with a control plasmid, or Strep-tagged wild-type FMDV 3Cpro, or 3Cpro C142T plasmid, together with a Flag-tagged porcine RIG-I plasmid in the absence or presence of Rupintrivir. (F) HEK293T cells were transiently transfected with Flag-tagged porcine RIG-I plasmid, control plasmid, or Strep-tagged wild-type FMDV 3Cpro or FMDV 3Cpro C142T plasmid, and subsequent treatment of Rupintrivir into…

FIGURE 6. Pathogenesis of the recombinant viruses in pigs. The experiment was carried out with six pigs which were divided into two groups (n = 3). (A) One group was infected with rShamir-3C-C142T (animals #32, #33, and #34) and the (B) other group was infected with rShamir-WT (animals #28, #29, and #31), intradermally at a concentration of 1 × 103.0 TCID50/0.1 mL. The right Y-axis of the graph shows the amount of virus in sera and swab as log10 values and the left Y-axis shows the clinical index. (C) percentage inhibition and (D,E) virus-neutralizing titer of each recombinant FMDV infected animals in two groups were measured.