3Cpro of FMDV inhibits type II interferon-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation

Abstract

Foot-and-mouth disease virus (FMDV) has developed various strategies to antagonize the host innate immunity. FMDV L^pro and 3C^pro interfere with type I IFNs through different mechanisms. The structural protein VP3 of FMDV degrades Janus kinase 1 to suppress IFN-γ signaling transduction. Whether non-structural proteins of FMDV are involved in restraining type II IFN signaling pathways is unknown. In this study, it was shown that FMDV replication was resistant to IFN-γ treatment after the infection was established and FMDV inhibited type II IFN induced expression of IFN-γ-stimulated genes (ISGs). We also showed for the first time that FMDV non-structural protein 3C antagonized IFN-γ-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation. 3C^pro expression significantly reduced the ISGs transcript levels and palindromic gamma-activated sequences (GAS) promoter activity, without affecting the protein level, tyrosine phosphorylation, and homodimerization of STAT1. Finally, we provided evidence that 3C protease activity played an essential role in degrading KPNA1 and thus inhibited ISGs mRNA and GAS promoter activities. Our results reveal a novel mechanism by which an FMDV non-structural protein antagonizes host type II IFN signaling.

Keywords: 3C; Foot-and-mouth disease virus (FMDV); IFN-γ; JAK-STAT signaling pathway; KPNA1; STAT1.

Fig. 1

The anti-FMDV activity of IFN-γ and inhibition of the expression of IFN-γ stimulated genes by FMDV. A PK-15 ​cells were separately pretreated with 0, 50, 100, 200, 400 ​ng/mL porcine IFN-γ for 5 ​h and then infected with FMDV O/BY/CHA/2010 ​at an MOI of 0.1. 24 h later, TCID₅₀ was determined on BHK-21 ​cells. B PK-15 ​cells infected with FMDV O/BY/CHA/2010 ​at an MOI of 0.1 for 24 ​h were treated with 0, 50, 100, 200, 400 ​ng/mL porcine IFN-γ. 5 h later, TCID₅₀ was determined on BHK-21 ​cells. C PK-15 ​cells were infected with FMDV O/BY/CHA/2010 ​at an MOI of 0.1 and growth kinetics was determined. PK-15 ​cells were inoculated with FMDV (D) or UV-inactivated FMDV (E) at an MOI of 0.1 for 24 ​h and then treated with porcine IFN-γ (200 ​ng/mL) for 5 ​h. The mRNA levels of poGBP2, poISG15, poISG56, poOAS and poPKR were examined using real-time RT-PCR. The levels of relative transcript were shown as relative fold changes, compared with the mock-treated control level of uninfected cells. Data represent the mean ​± ​the standard deviations (error bars) of three independent experiments. ∗, P ​< ​0.05.

Fig. 2

FMDV 3C^pro suppressed ISGs mRNA synthesis and GAS promoter activity of type II IFN signaling. A HeLa cells transfected with pXJ41, pXJ41-GST, pXJ41-3ABC, pXJ41-3A, pXJ41-3B or pXJ41-3C were treated with 100 ​ng/mL of human IFN-γ at 24 ​h after transfection. 5 h later, the ISGs transcript levels were measured by real-time RT-PCR, with pXJ41 and pXJ41-GST as negative controls. The levels of relative transcript were shown as relative fold changes, compared with the mock-treated control level of uninfected cells. Data represent the mean ​± ​the standard deviations (error bars) of three independent experiments. B Expression of 3ABC, 3A, 3B or 3C protein was detected by Western blotting using antibody against FLAG or antibody against HA. β-actin was used as a protein loading control. C HeLa cells grown in 12-well plates were cotransfected with 0 ​μg, 0.125 ​μg, 0.25 ​μg or 0.5 ​μg of pXJ41, pXJ41-GST, pXJ41-3ABC, pXJ41-3A, pXJ41-3B or pXJ41-3C and 0.5 ​μg of pGAS-Luc as well as 0.05 ​μg of pRL-TK as an internal control. pXJ41 and pXJ41-GST were worked as negative controls. At 24 ​h after transfection, cells were treated with 100 ​ng/mL of human IFN-γ. 16 h later, cells were lysed and reporter expressions were measured using the Dual-Luciferase reporter assay kit (Promega). The values were normalized regarding Renilla luciferase activities. Then relative expression levels were calculated and shown as relative fold changes, compared with the mock-treated control of untransfected cells. Data represent the mean ​± ​the standard deviations (error bars) of three independent experiments. D Expression of 3ABC, 3A, 3B or 3C protein was detected by Western blotting using antibody against FLAG or antibody against HA. β-actin was used as a protein loading control. ∗, P ​< ​0.05.

Fig. 3

The catalytic triad H46, D84 and C163 of 3C^pro played essential roles in suppressing ISGs mRNA synthesis and GAS promoter activity of type II IFN signaling. A HeLa cells transfected with pXJ41, pXJ41-3C or individual mutants of pXJ41-3C were treated with 100 ​ng/mL human IFN-γ at 24 ​h after transfection. 5 h later, the hGBP1, hISG15, hISG56, hOAS and hPKR transcript levels were analyzed by real-time RT-PCR as described above. Data represent the means ​± ​the standard deviations (error bars) of three independent experiments, with each experiment in triplicate. B Expression of 3C or individual mutant protein was detected by Western blotting using antibody against HA. β-actin was used as a protein loading control. C HeLa cells transfected with 0.5 ​μg of pXJ41, pXJ41-3C or its individual mutants of pXJ41-3C and 0.5 ​μg of pGAS-Luc as well as 0.05 ​μg of pRL-TK. At 24 ​h after transfection, cells were treated with 100 ​ng/mL human IFN-γ. 16 h later, cells were lysed and reporter expressions were measured using the Dual-Luciferase reporter assay kit as described above. Data represent the mean ​± ​the standard deviations (error bars) of three independent experiments. D Expression of 3C or individual mutant protein in HeLa cells transfected with indicated plasmid was detected by Western blotting using antibody against HA. β-actin was used as a protein loading control. ∗, P ​< ​0.05.

Fig. 4

STAT1 expression level, phosphorylation status and its homodimer formation in FMDV 3C^pro-expressing cells after IFN-γ stimulation. A HeLa cells transfected with pXJ41 or pXJ41-3C were treated with 100 ​ng/mL human IFN-γ for 1 ​h at 24 ​h after transfection and collected for Western blotting with antibody against STAT1 (top panel), phospho-STAT1 (STAT1-Y701, panel 2) or HA (panel 3), respectively. Antibody against β-actin (bottom panel) was used as a protein loading control. B Densitometric analysis of the digital image in (A). Intensities of the band were normalized with that of β-actin. C Cells transfected with pXJ41 or pXJ41-3C were treated with human 100 ​ng/mL IFN-γ for 1 ​h at 24 ​h post-transfection. Samples were lysed and subjected to native-PAGE and immunoblotting with antibody against STAT1 (top panel). Expression of 3C^pro was detected by SDS-PAGE and immunoblotting with antibody against HA (panel 2), with β-actin as a loading control (bottom panel). D Densitometric analysis of the digital image in (C). Intensities of the band were normalized with that of β-actin. Data represent the mean ​± ​the standard deviations (error bars) of three independent experiments.

Fig. 5

FMDV 3C^pro inhibits nuclear translocation of STAT1 as directed by IFA. HeLa cells transfected with pXJ41, pXJ41-3C, or individual mutants of pXJ41-3C were either left untreated (−) or treated (+) with human 100 ​ng/mL IFN-γ for 1 ​h at 18 ​h post-transfection. Cells were fixed, and an IFA was conducted. Yellow arrows indicate WT or mutated 3C^pro-expressing cells, and white arrows indicate untransfected cells. Scale bar, 10 ​μm.

Fig. 6

Phosphorylated STAT1 in nucleus and cytoplasm. A Western blot analysis of phosphorylated STAT1 in nucleus and cytoplasm fraction. HeLa cells were performed for nuclear and cytoplasmic separation after human IFN-γ treatment for 1 ​h. Samples were analyzed by Western blotting using antibody against STAT1-Y701 (top panel), antibodies against HSP90 and PARP were used as cytoplasmic and nuclear protein marker respectively. 3C^pro expression of FMDV was detected using antibody against HA (panel 4), with antibody against β-actin as a loading control (bottom panel). B Densitometric analysis of the band of phosphorylated STAT1 in nucleus and cytoplasm. Intensities of the band were shown as the relative percentage of the total density of their counterpart, cytoplasmic and nuclear fractions normalized with HSP90 and PARP respectively. Data represent the mean ​± ​the standard deviations (error bars) of three experiments. -: without IFN-γ treatment, +: with IFN-γ treatment. ∗, P ​< ​0.05.

Fig. 7

The antiviral activity of IFN-γ against FMDV was recovered by KPNA1 overexpression. A PK-15 ​cells infected with FMDV O/BY/CHA/2010 ​at an MOI of 0.1 for 2 ​h were transfected with 0 ​μg, 0.25 ​μg, 0.5 ​μg or 0.75 ​μg of pXJ41-FLAG-KPNA1. Empty vector pXJ41 was used to ensure that the total amount of transfected plasmid DNA in each group was 0.75 ​μg. At 24 ​h after infection, cells were treated with 400 ​ng/mL porcine IFN-γ. 5 h later, TCID₅₀ was determined on BHK-21 ​cells. Data represent the mean ​± ​the standard deviations (error bars) of three experiments. B Expression of KPNA1 (top panel) or FMDV 3C^pro (middle panel) in PK-15 ​cells detected by Western blotting using antibody against FLAG or rabbit 3C serum, with β-actin as a loading control (bottom panel). C Densitometric analysis of the digital image of FMDV 3C^pro. The band intensities normalized with β-actin were suggested as relative protein expression. Data represent the mean ​± ​the standard deviations (error bars) of three experiments. ∗, P ​< ​0.05.

Fig. 8

ISGs mRNA level of type II IFN signaling is associated with FMDV 3C protease activity in degrading KPNA1. HeLa cells cotransfected with increased dose of pXJ41-FLAG-KPNA1 and pXJ41-3C or its mutants were treated with 100 ​-ng/mL human IFN-γ at 24 ​h post-transfection. 5 h later, the mRNA levels of hGBP1 (A), hISG15 (B), hISG56 (C), hOAS (D) and hPKR (E) were detected by real-time RT-PCR as described above. Data represent the mean ​± ​the standard deviations (error bars) of three independent experiments. F Expression of KPNA1, 3C or its mutant protein was measured by Western blotting using antibody against FLAG or HA. β-actin was used as a protein loading control. ∗, P ​< ​0.05.

Fig. 9

GAS promoter activity of type II IFN signaling is associated with FMDV 3C protease activity in degrading KPNA1. A HeLa cells cotransfected with increased dose of pXJ41-FLAG-KPNA1 and pXJ41-3C or its mutants and pGAS-Luc as well as pRL-TK for 24 ​h were incubated with 100 ​ng/mL human IFN-γ for 16 ​h. Luciferase assay was performed as described above. Data represent the mean ​± ​the standard deviations (error bars) of three experiments. B Expression of KPNA1, 3C or its mutant protein was measured by Western blotting using antibody against FLAG or HA. β-actin was used as a protein loading control. ∗, P ​< ​0.05.