Author Contributions
J.T. and L.Q. conceived and designed the experiments; H.K., X.H., and X.Z. performed the experiments; H.K., D.L., C.L., and H.W. analyzed the data; D.L., D.G., J.L., and Q.J. contributed reagents/materials/analysis tools; H.K., H.Y., and J.L. wrote the paper. D.L., H.K., and X.H. revised the paper.
Figure 1.
Effect of feline panleucopenia virus (FPV) on interferon (IFN)-β production and Sendai virus (SeV)-mediated IFN-β production as indicated by luciferase activity in F81 cells. The cells in this experiment were co-transfected with IFN-β-Luc and the Renilla luciferase construct pRL-TK, followed by a mock infection or infection with FPV. After 12 h of co-transfection, the cells were stimulated with SeV (SeV+) or left untreated (SeV-). The data represent the relative firefly luciferase activity normalized to the Renilla luciferase activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups. The FPV infection was monitored by immunoblotting using a mouse anti-capsid protein 2 (VP2) antibody, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.
Figure 1.
Effect of feline panleucopenia virus (FPV) on interferon (IFN)-β production and Sendai virus (SeV)-mediated IFN-β production as indicated by luciferase activity in F81 cells. The cells in this experiment were co-transfected with IFN-β-Luc and the Renilla luciferase construct pRL-TK, followed by a mock infection or infection with FPV. After 12 h of co-transfection, the cells were stimulated with SeV (SeV+) or left untreated (SeV-). The data represent the relative firefly luciferase activity normalized to the Renilla luciferase activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups. The FPV infection was monitored by immunoblotting using a mouse anti-capsid protein 2 (VP2) antibody, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.
Figure 2.
FPV NS2 as a negative regulator impedes SeV-mediated IFN-β induction. (A) Effects of protein-coding genes of FPV on the SeV-induced IFN-β promoter activation in F81 cells. The cells in this experiment were co-transfected with IFN-β-Luc, the Renilla luciferase construct pRL-TK and one of the recombinant plasmids pFlag-vp1, pFlag-vp2, pFlag-ns1, or pFlag-ns2. Twenty-four hours later, the cells were stimulated with SeV. The luciferase activity was measured at 12 h after simulation. The values were normalized to the Renilla activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and “*” indicates significant differences (p < 0.05) between groups. The expression of VP1, VP2, NS1, or NS2 was monitored by immunoblotting using a mouse anti-Flag antibody; GAPDH was used as a loading control. (B) The SeV-mediated IFN-β expression is disrupted by NS2. F81 cells were transfected with p3×Flag-NS2. At 12 h post transfection, the cells were inoculated with SeV. The cell lysates at 0, 6, 12 and 24 h after SeV infection were analyzed by immunoblotting (IB) using anti-Flag and anti-IFN-β antibodies. (C). NS2 inhibits IFN promoter activity in a dose-dependent manner. F81 cells were co-transfected with IFN-β-Luc, pRL-TK and different amounts of p3×Flag-NS2 (10, 50, 100, 200 or 400 ng). At 12 h post transfection, the cells were inoculated with SeV. Twelve hours after infection, the cells were harvested, and the luciferase activities were measured. The values were normalized to the Renilla activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups.
Figure 2.
FPV NS2 as a negative regulator impedes SeV-mediated IFN-β induction. (A) Effects of protein-coding genes of FPV on the SeV-induced IFN-β promoter activation in F81 cells. The cells in this experiment were co-transfected with IFN-β-Luc, the Renilla luciferase construct pRL-TK and one of the recombinant plasmids pFlag-vp1, pFlag-vp2, pFlag-ns1, or pFlag-ns2. Twenty-four hours later, the cells were stimulated with SeV. The luciferase activity was measured at 12 h after simulation. The values were normalized to the Renilla activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and “*” indicates significant differences (p < 0.05) between groups. The expression of VP1, VP2, NS1, or NS2 was monitored by immunoblotting using a mouse anti-Flag antibody; GAPDH was used as a loading control. (B) The SeV-mediated IFN-β expression is disrupted by NS2. F81 cells were transfected with p3×Flag-NS2. At 12 h post transfection, the cells were inoculated with SeV. The cell lysates at 0, 6, 12 and 24 h after SeV infection were analyzed by immunoblotting (IB) using anti-Flag and anti-IFN-β antibodies. (C). NS2 inhibits IFN promoter activity in a dose-dependent manner. F81 cells were co-transfected with IFN-β-Luc, pRL-TK and different amounts of p3×Flag-NS2 (10, 50, 100, 200 or 400 ng). At 12 h post transfection, the cells were inoculated with SeV. Twelve hours after infection, the cells were harvested, and the luciferase activities were measured. The values were normalized to the Renilla activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups.
Figure 3.
Inhibition of the activity of transcription factors on specific PRDs of the IFN-β promoter by NS2. F81 cells were co-transfected with p3×Flag-NS2, pRL-TK and either p3×PRDII-Luc (A), PRDIII/I-Luc (B), or p6×PRDIV-Luc (C). At 12 h post-transfection, the cells were inoculated with SeV. Twelve hours after infection, the cells were harvested and the luciferase activities were measured. The values were normalized to the Renilla activity. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups. (D) The phosphorylation levels of IRF3 in the cells stimulated with SeV after transfection with NS2. F81 cells were transfected with p3×Flag-NS2. At 12 h post-transfection, the cells were inoculated with SeV. The cell lysates at 0, 3, 6, 9 and 12 h after SeV infection were analyzed by immunoblotting using antibodies against IRF3 or phosphorylated IRF3 and the quantification of relative p-IRF3 band intensities to IRF3 was showed with histogram.
Figure 3.
Inhibition of the activity of transcription factors on specific PRDs of the IFN-β promoter by NS2. F81 cells were co-transfected with p3×Flag-NS2, pRL-TK and either p3×PRDII-Luc (A), PRDIII/I-Luc (B), or p6×PRDIV-Luc (C). At 12 h post-transfection, the cells were inoculated with SeV. Twelve hours after infection, the cells were harvested and the luciferase activities were measured. The values were normalized to the Renilla activity. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups. (D) The phosphorylation levels of IRF3 in the cells stimulated with SeV after transfection with NS2. F81 cells were transfected with p3×Flag-NS2. At 12 h post-transfection, the cells were inoculated with SeV. The cell lysates at 0, 3, 6, 9 and 12 h after SeV infection were analyzed by immunoblotting using antibodies against IRF3 or phosphorylated IRF3 and the quantification of relative p-IRF3 band intensities to IRF3 was showed with histogram.
Figure 4.
FPV NS2 blocks interferon induction at a step upstream of IRF3. F81 cells were co-transfected with IRF3-Luc, pRL-TK, p3×Flag-NS2 and a plasmid expressing one of the molecules in the IRF3 signaling pathway: MAVS (A), STING (B), TBK1 (C), and IRF3-5D (D), respectively. The cell lysates were used to measure the firefly and Renilla luciferase activities at 24 h post-transfection. The values were normalized to the Renilla activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups.
Figure 4.
FPV NS2 blocks interferon induction at a step upstream of IRF3. F81 cells were co-transfected with IRF3-Luc, pRL-TK, p3×Flag-NS2 and a plasmid expressing one of the molecules in the IRF3 signaling pathway: MAVS (A), STING (B), TBK1 (C), and IRF3-5D (D), respectively. The cell lysates were used to measure the firefly and Renilla luciferase activities at 24 h post-transfection. The values were normalized to the Renilla activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups.
Figure 5.
Analysis of the interaction between NS2 and TBK1. (A) Flag-tagged NS2 was co-transfected with Myc-tagged TBK1 into F81 cells for 36 h. The cell lysates were immunoprecipitated using ANTI-FLAG M2 magnetic beads. The whole-cell lysates and immunoprecipitation complexes were analyzed by immunoblotting using anti-Flag or anti-Myc antibodies. (B) F81 cells were co-transfected with the pDsred-TBK1 plasmid and pEGFP-NS2 plasmid. Twenty-four hours after transfection, the nuclei were stained with DAPI. The fluorescent images of the cells were obtained with a confocal laser scanning microscope.
Figure 5.
Analysis of the interaction between NS2 and TBK1. (A) Flag-tagged NS2 was co-transfected with Myc-tagged TBK1 into F81 cells for 36 h. The cell lysates were immunoprecipitated using ANTI-FLAG M2 magnetic beads. The whole-cell lysates and immunoprecipitation complexes were analyzed by immunoblotting using anti-Flag or anti-Myc antibodies. (B) F81 cells were co-transfected with the pDsred-TBK1 plasmid and pEGFP-NS2 plasmid. Twenty-four hours after transfection, the nuclei were stained with DAPI. The fluorescent images of the cells were obtained with a confocal laser scanning microscope.
Figure 6.
NS2 disrupts the TBK1–STING interaction and reduces the phosphorylation of STING and downstream IRF3. (A) F81 cells were transfected with Flag-tagged STING, Myc-tagged TBK1 and V5-tagged NS2 for 36 h. The cell lysates were subjected to immunoprecipitation with ANTI-FLAG M2 magnetic beads. The whole-cell lysates and IP complexes were analyzed by western blot using anti-Flag or anti-Myc antibodies. (B) F81 cells were transfected with Flag-tagged STING and V5-tagged NS2 for 36 h. The cell lysates were resolved by SDS-PAGE, after which they were analyzed by western blot with antibodies against STING or phosphorylated STING. (C) F81 cells were transfected with Flag-tagged NS2. Twenty-four hours later, the cells were stimulated with SeV. The cytoplasmic and nuclear extracts of the F81 cells were separated at 6 h after simulation and analyzed by western blot with antibodies against IRF3 or phosphorylated IRF3 and the quantification of relative p-IRF3 band intensities to IRF3 was showed with histogram.
Figure 6.
NS2 disrupts the TBK1–STING interaction and reduces the phosphorylation of STING and downstream IRF3. (A) F81 cells were transfected with Flag-tagged STING, Myc-tagged TBK1 and V5-tagged NS2 for 36 h. The cell lysates were subjected to immunoprecipitation with ANTI-FLAG M2 magnetic beads. The whole-cell lysates and IP complexes were analyzed by western blot using anti-Flag or anti-Myc antibodies. (B) F81 cells were transfected with Flag-tagged STING and V5-tagged NS2 for 36 h. The cell lysates were resolved by SDS-PAGE, after which they were analyzed by western blot with antibodies against STING or phosphorylated STING. (C) F81 cells were transfected with Flag-tagged NS2. Twenty-four hours later, the cells were stimulated with SeV. The cytoplasmic and nuclear extracts of the F81 cells were separated at 6 h after simulation and analyzed by western blot with antibodies against IRF3 or phosphorylated IRF3 and the quantification of relative p-IRF3 band intensities to IRF3 was showed with histogram.
Figure 7.
The C-terminus plus the coiled coil domain of NS2 can inhibit the IRF3 signaling pathway. (A) Schematic diagram of the truncated NS2 constructs. (B) F81 cells were co-transfected with IRF3-Luc, pRL-TK and plasmids expressing either the intact NS2, the N-terminal domain of NS2, the C-terminal domain of NS2 or the C-terminal domain plus the coiled coil domain of NS2. The effect of each truncated NS2 on the expression of the luciferase gene under the control of the IRF3 binding region PRDIII/I of the feline IFN-β promoter was determined using a luciferase assay. The values were normalized to the Renilla activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups. The expression of the intact NS2, the N-terminal domain of NS2, the C-terminal domain of NS2 or the C-terminal domain plus the coiled coil domain of NS2 was monitored by immunoblotting using a mouse anti-Flag antibody; GAPDH was used as a loading control.
Figure 7.
The C-terminus plus the coiled coil domain of NS2 can inhibit the IRF3 signaling pathway. (A) Schematic diagram of the truncated NS2 constructs. (B) F81 cells were co-transfected with IRF3-Luc, pRL-TK and plasmids expressing either the intact NS2, the N-terminal domain of NS2, the C-terminal domain of NS2 or the C-terminal domain plus the coiled coil domain of NS2. The effect of each truncated NS2 on the expression of the luciferase gene under the control of the IRF3 binding region PRDIII/I of the feline IFN-β promoter was determined using a luciferase assay. The values were normalized to the Renilla activity. The data represent the mean values of three independent experiments. The error bars represent standard deviations, and the asterisks indicate significant differences (*: p < 0.05; **: p < 0.01; ***: p < 0.001) between groups. The expression of the intact NS2, the N-terminal domain of NS2, the C-terminal domain of NS2 or the C-terminal domain plus the coiled coil domain of NS2 was monitored by immunoblotting using a mouse anti-Flag antibody; GAPDH was used as a loading control.