Figure 1.
Porcine E3 ubiquitin ligase RNF122 was up-regulated after PRRSV infection. (a) Heat map of differential expression of E3 ubiquitin ligase in RING domain family after PRRSV infection. (b) 3D4/21 cells were infected with PRRSV (MOI = 0.5), and the cells were harvested at different time points for qRT-PCR detection to analyze the expression of RNF122. (c) 3D4/21 cells were infected with PRRSV (MOI = 0.5), and (d) the expression of RNF122 was observed by fluorescence microscopy at different time points. * p < 0.05 (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 1.
Porcine E3 ubiquitin ligase RNF122 was up-regulated after PRRSV infection. (a) Heat map of differential expression of E3 ubiquitin ligase in RING domain family after PRRSV infection. (b) 3D4/21 cells were infected with PRRSV (MOI = 0.5), and the cells were harvested at different time points for qRT-PCR detection to analyze the expression of RNF122. (c) 3D4/21 cells were infected with PRRSV (MOI = 0.5), and (d) the expression of RNF122 was observed by fluorescence microscopy at different time points. * p < 0.05 (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 2.
The core promoter region of porcine RNF22 was identified. (a) Schematic diagram of constitution of luciferase mutant plasmid. (b) HEK293 T cells were co-transfected with Luciferase mutant plasmid and RL-TK, luciferase activity was measured using the supernatant of lysate cells. (c) HEK293 T cells were co-transfected with pTP1-Luc, pTP6-Luc and RL-TK, infected with PRRSV (MOI = 0.5) or not, luciferase activity was measured using the supernatant of lysate cells. pGL3-Basic was the negative control and pGL3-Control was the positive control. ** p < 0.01; *** p < 0.001 ns, no significant difference (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 2.
The core promoter region of porcine RNF22 was identified. (a) Schematic diagram of constitution of luciferase mutant plasmid. (b) HEK293 T cells were co-transfected with Luciferase mutant plasmid and RL-TK, luciferase activity was measured using the supernatant of lysate cells. (c) HEK293 T cells were co-transfected with pTP1-Luc, pTP6-Luc and RL-TK, infected with PRRSV (MOI = 0.5) or not, luciferase activity was measured using the supernatant of lysate cells. pGL3-Basic was the negative control and pGL3-Control was the positive control. ** p < 0.01; *** p < 0.001 ns, no significant difference (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 3.
E2F complex and HLTF were identified as key transcription factors in porcine RNF122. (a) Schematic diagram of transcription factor binding site mutation in RNF122 core promoter region. (b) HEK293T cells were co-transfected with mutant plasmids of pTP6-Luc and RL-TK, luciferase activity was measured using the supernatant of lysate cells. pGL3-Basic was the negative control and pGL3-Control was the positive control. (c) 3D4/21 cells were infected with PRRSV (MOI = 0.5), and the cells were collected 24 h later for qRT-PCR detection to analyze the expression of transcription factors. ** p < 0.01; *** p < 0.001; ns, no significant difference; N.D., no determined. (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 3.
E2F complex and HLTF were identified as key transcription factors in porcine RNF122. (a) Schematic diagram of transcription factor binding site mutation in RNF122 core promoter region. (b) HEK293T cells were co-transfected with mutant plasmids of pTP6-Luc and RL-TK, luciferase activity was measured using the supernatant of lysate cells. pGL3-Basic was the negative control and pGL3-Control was the positive control. (c) 3D4/21 cells were infected with PRRSV (MOI = 0.5), and the cells were collected 24 h later for qRT-PCR detection to analyze the expression of transcription factors. ** p < 0.01; *** p < 0.001; ns, no significant difference; N.D., no determined. (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 4.
Transcriptional regulations of RNF122 were mediated by PRRSV nsp1α, nsp7 and nsp9. (a) HEK293T cells were co-transfected with pTP6-Luc and pRL-TK, and then transfected nsp1α, nsp1β, nsp4, nsp5, nsp7, nsp9, nsp10, nsp11, N plasmids, respectively, or infected with PRRSV (MOI = 0.5), luciferase activity was measured using the supernatant of lysate cells. pGL3-Basic was the negative control and pGL3-Control was the positive control. (b) HEK293T cells were co-transfected with pTP6-Luc and RL-TK, transfected with nsp9 or not, luciferase activity was measured using the supernatant of lysate cells. (c) 3D4/21 cells were infected with nsp9 or not, and the cells were collected 24 h later for qRT-PCR detection to analyze the expression of RNF122 and RB. (d) 3D4/21 cells were infected with PRRSV (MOI = 0.5), or transfected nsp1α, nsp7 or not, and the cells were collected 24 h later for qRT-PCR detection to analyze the expression of RNF122 and HLTF. (e) 3D4/21 cells were infected with PRRSV (MOI = 0.5), or transfected nsp1α, nsp7 or not, and the cells were collected 24 h later to analyze the protein expression of HLTF. *** p < 0.001; **** p < 0.0001 (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 4.
Transcriptional regulations of RNF122 were mediated by PRRSV nsp1α, nsp7 and nsp9. (a) HEK293T cells were co-transfected with pTP6-Luc and pRL-TK, and then transfected nsp1α, nsp1β, nsp4, nsp5, nsp7, nsp9, nsp10, nsp11, N plasmids, respectively, or infected with PRRSV (MOI = 0.5), luciferase activity was measured using the supernatant of lysate cells. pGL3-Basic was the negative control and pGL3-Control was the positive control. (b) HEK293T cells were co-transfected with pTP6-Luc and RL-TK, transfected with nsp9 or not, luciferase activity was measured using the supernatant of lysate cells. (c) 3D4/21 cells were infected with nsp9 or not, and the cells were collected 24 h later for qRT-PCR detection to analyze the expression of RNF122 and RB. (d) 3D4/21 cells were infected with PRRSV (MOI = 0.5), or transfected nsp1α, nsp7 or not, and the cells were collected 24 h later for qRT-PCR detection to analyze the expression of RNF122 and HLTF. (e) 3D4/21 cells were infected with PRRSV (MOI = 0.5), or transfected nsp1α, nsp7 or not, and the cells were collected 24 h later to analyze the protein expression of HLTF. *** p < 0.001; **** p < 0.0001 (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 5.
Porcine RNF122 promoted viral replication of PRRSV. (a,b) 3D4/21 cells were transfected with Flag-CMV or Flag-RNF122, 12 h later, cells were infected with PRRSV (MOI = 0.5). mRNA loads of PRRSV N and nsp2 were tested 12 h later by qRT-PCR. (c) 3D4/21 cells were transfected with Flag-CMV or Flag-RNF122, 12 h later, cells were infected with PRRSV (MOI = 0.5). PRRSV nsp2 were 12 h later tested by WB. (d,e) 3D4/21 cells were transfected with SiRNA or not; 12 h later, cells were infected with PRRSV (MOI = 0.5). mRNA loads of PRRSV nsp2 and RNF122 were tested 12 h later by qRT-PCR. (f) 3D4/21 cells were transfected with SiRNA or not; 12 h later, cells were infected with PRRSV (MOI = 0.5). PRRSV nsp2 was tested 12 h later by WB. (g) 3D4/21 cells were transfected with RNF122 or SiRNA; 12 h later, cells were infected with PRRSV (MOI = 0.5). PRRSV-N genes as an indicator to represent virus copy number and viral copies of PRRSV were tested 12 h later by qRT-PCR. (h) 3D4/21 cells were transfected with RNF122 or SiRNA; 12 h later, cells were infected with PRRSV (MOI = 0.5). TCID50 was calculated 48 h later by Reed Muench method. (i) 3D4/21 cells were transfected with RNF122 or SiRNA; 12 h later, cells were infected with PRRSV (MOI = 0.5). RNF122 was checked 12 h later by WB. *** p < 0.001 (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 5.
Porcine RNF122 promoted viral replication of PRRSV. (a,b) 3D4/21 cells were transfected with Flag-CMV or Flag-RNF122, 12 h later, cells were infected with PRRSV (MOI = 0.5). mRNA loads of PRRSV N and nsp2 were tested 12 h later by qRT-PCR. (c) 3D4/21 cells were transfected with Flag-CMV or Flag-RNF122, 12 h later, cells were infected with PRRSV (MOI = 0.5). PRRSV nsp2 were 12 h later tested by WB. (d,e) 3D4/21 cells were transfected with SiRNA or not; 12 h later, cells were infected with PRRSV (MOI = 0.5). mRNA loads of PRRSV nsp2 and RNF122 were tested 12 h later by qRT-PCR. (f) 3D4/21 cells were transfected with SiRNA or not; 12 h later, cells were infected with PRRSV (MOI = 0.5). PRRSV nsp2 was tested 12 h later by WB. (g) 3D4/21 cells were transfected with RNF122 or SiRNA; 12 h later, cells were infected with PRRSV (MOI = 0.5). PRRSV-N genes as an indicator to represent virus copy number and viral copies of PRRSV were tested 12 h later by qRT-PCR. (h) 3D4/21 cells were transfected with RNF122 or SiRNA; 12 h later, cells were infected with PRRSV (MOI = 0.5). TCID50 was calculated 48 h later by Reed Muench method. (i) 3D4/21 cells were transfected with RNF122 or SiRNA; 12 h later, cells were infected with PRRSV (MOI = 0.5). RNF122 was checked 12 h later by WB. *** p < 0.001 (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 6.
Interaction between porcine RNF122 and PRRSV nsp4. (a) HEK293T cells were co-transfected with Myc-RNF122 and Flag-nsp1α, Flag-nsp4, Flag-nsp5, Flag-nsp7, Flag-nsp9, Flag-nsp10, Flag-nsp11, Flag-N or Flag-CMV. Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-Flag antibody and an anti-Myc antibody, respectively. Red arrow represents interaction bands. (b) 3D4/21 cells were placed on cover slips in 12-well plates and co-transfected with the Myc-RNF122 and Flag-nsp4 or Flag-CMV, respectively. Fixed double-stained with a mouse anti-Myc mAb and a rabbit anti-Flag antibody, and attended by FITC-conjugated anti-mouse IgG (green) and PE-conjugated anti-rabbit IgG (red). Nuclei were stained with DAPI (blue). Fluorescence confocal microscopy (UltraView Vox, PerkinElmer, Waltham, MA, USA) was used to detect the co-location. (c) HEK293T cells were co-transfected with Flag-nsp4 (1 μg) and Myc-RNF122 (0 μg, 0.1 μg, 0.5 μg, 1 μg), 24 h later, the protein expression was tested by WB.
Figure 6.
Interaction between porcine RNF122 and PRRSV nsp4. (a) HEK293T cells were co-transfected with Myc-RNF122 and Flag-nsp1α, Flag-nsp4, Flag-nsp5, Flag-nsp7, Flag-nsp9, Flag-nsp10, Flag-nsp11, Flag-N or Flag-CMV. Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-Flag antibody and an anti-Myc antibody, respectively. Red arrow represents interaction bands. (b) 3D4/21 cells were placed on cover slips in 12-well plates and co-transfected with the Myc-RNF122 and Flag-nsp4 or Flag-CMV, respectively. Fixed double-stained with a mouse anti-Myc mAb and a rabbit anti-Flag antibody, and attended by FITC-conjugated anti-mouse IgG (green) and PE-conjugated anti-rabbit IgG (red). Nuclei were stained with DAPI (blue). Fluorescence confocal microscopy (UltraView Vox, PerkinElmer, Waltham, MA, USA) was used to detect the co-location. (c) HEK293T cells were co-transfected with Flag-nsp4 (1 μg) and Myc-RNF122 (0 μg, 0.1 μg, 0.5 μg, 1 μg), 24 h later, the protein expression was tested by WB.
Figure 7.
RNF122 performed K63-linked ubiquitination lysine of PRRSV nsp4 at position 170. (a) HEK293T cells were co-transfected with Myc-RNF122, Flag-nsp4 and HA-Ub (WT) or HA-Ub (K63). Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively. (b) HEK293T cells were co-transfected with SiRNF122, Flag-nsp4 and HA-Ub (WT) or HA-Ub (K63). Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-RNF122 antibody, respectively. (c) HEK293T cells were co-transfected with Myc-RNF122, HA-Ub (WT) and Flag-nsp4, Flag-nsp4(K7R), Flag-nsp4(K33R), Flag-nsp4(K79R), Flag-nsp4(K158R), Flag-nsp4(K170R) or not. Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively.
Figure 7.
RNF122 performed K63-linked ubiquitination lysine of PRRSV nsp4 at position 170. (a) HEK293T cells were co-transfected with Myc-RNF122, Flag-nsp4 and HA-Ub (WT) or HA-Ub (K63). Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively. (b) HEK293T cells were co-transfected with SiRNF122, Flag-nsp4 and HA-Ub (WT) or HA-Ub (K63). Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-RNF122 antibody, respectively. (c) HEK293T cells were co-transfected with Myc-RNF122, HA-Ub (WT) and Flag-nsp4, Flag-nsp4(K7R), Flag-nsp4(K33R), Flag-nsp4(K79R), Flag-nsp4(K158R), Flag-nsp4(K170R) or not. Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively.
Figure 8.
Porcine RNF122 negatively regulated type I interferon signaling pathway. (a,b) 3D4/21 cells were transfected with Flag-CMV or Flag-RNF122, 24 h later, mRNA loads of IFNβ and NF-κB were tested by qRT-PCR. (c) 3D4/21 cells were transfected with Flag-CMV or Flag-RNF122, 24 h later, RNF122 was tested by WB. (d–f) 3D4/21 cells were transfected with SiRNA or not, 24 h later, mRNA loads of IFN-β, NF-κB and RNF122 were tested by qRT-PCR. (g) 3D4/21 cells were transfected with SiRNA or not, 24 h later, RNF122 was tested by WB. ** p < 0.01; *** p < 0.001 (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 8.
Porcine RNF122 negatively regulated type I interferon signaling pathway. (a,b) 3D4/21 cells were transfected with Flag-CMV or Flag-RNF122, 24 h later, mRNA loads of IFNβ and NF-κB were tested by qRT-PCR. (c) 3D4/21 cells were transfected with Flag-CMV or Flag-RNF122, 24 h later, RNF122 was tested by WB. (d–f) 3D4/21 cells were transfected with SiRNA or not, 24 h later, mRNA loads of IFN-β, NF-κB and RNF122 were tested by qRT-PCR. (g) 3D4/21 cells were transfected with SiRNA or not, 24 h later, RNF122 was tested by WB. ** p < 0.01; *** p < 0.001 (analysis of two-way ANOVA attended by Bonferroni post-test). Data are representative of three independent experiments.
Figure 9.
Interaction between porcine RNF122 and MDA5 molecules. (a) HEK293T cells were co-transfected with Flag-RNF122, 24 h after transfection, Flag Beads were used for protein enrichment, and further detected by Western blotting. Then the gel was stained with silver using a ProteoSilver Silver Stain Kit (Solarbio Life Sciences). (b) HEK293T cells were co-transfected with HA-RNF122 and Flag-RIG-I, Flag-MDA5 or Flag-CMV. Flag Beads were used for CO-24 h after transfection IP, and further detected by Western blotting with an anti-Flag antibody and an anti-HA antibody, respectively. (c) HeLa cells were placed on coverslips in 12-well plates and co-transfected with the HA-RNF122 and Flag-RIG-I, Flag-MDA5 or Flag-CMV, respectively. Fixed double-stained with a mouse anti-HA mAb and a rabbit anti-Flag antibody, and followed by FITC-conjugated anti-mouse IgG (green) and PE-conjugated anti-rabbit IgG (red). Nuclei were stained with DAPI (blue). Fluorescence confocal microscopy (UltraView Vox, PerkinElmer, Waltham, MA, USA) was used to detect the co-location. (d) HEK293T cells were co-transfected with HA-RNF122 and Flag-MDA5, Flag-MDA5 (N), Flag-MDA5 (M), Flag-MDA5 (C)or Flag-CMV. Flag Beads were used for CO-IP 24 h after transfection, and further detected by Western blotting with an anti-Flag antibody and an anti-HA antibody, respectively. (e) HEK293T cells were co-transfected with Flag-MDA5 and Myc-RNF122, Myc-RNF122 (TM), Myc-RNF122 (RING) or Myc-CMV. Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-Flag antibody and an anti-Myc antibody, respectively.
Figure 9.
Interaction between porcine RNF122 and MDA5 molecules. (a) HEK293T cells were co-transfected with Flag-RNF122, 24 h after transfection, Flag Beads were used for protein enrichment, and further detected by Western blotting. Then the gel was stained with silver using a ProteoSilver Silver Stain Kit (Solarbio Life Sciences). (b) HEK293T cells were co-transfected with HA-RNF122 and Flag-RIG-I, Flag-MDA5 or Flag-CMV. Flag Beads were used for CO-24 h after transfection IP, and further detected by Western blotting with an anti-Flag antibody and an anti-HA antibody, respectively. (c) HeLa cells were placed on coverslips in 12-well plates and co-transfected with the HA-RNF122 and Flag-RIG-I, Flag-MDA5 or Flag-CMV, respectively. Fixed double-stained with a mouse anti-HA mAb and a rabbit anti-Flag antibody, and followed by FITC-conjugated anti-mouse IgG (green) and PE-conjugated anti-rabbit IgG (red). Nuclei were stained with DAPI (blue). Fluorescence confocal microscopy (UltraView Vox, PerkinElmer, Waltham, MA, USA) was used to detect the co-location. (d) HEK293T cells were co-transfected with HA-RNF122 and Flag-MDA5, Flag-MDA5 (N), Flag-MDA5 (M), Flag-MDA5 (C)or Flag-CMV. Flag Beads were used for CO-IP 24 h after transfection, and further detected by Western blotting with an anti-Flag antibody and an anti-HA antibody, respectively. (e) HEK293T cells were co-transfected with Flag-MDA5 and Myc-RNF122, Myc-RNF122 (TM), Myc-RNF122 (RING) or Myc-CMV. Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-Flag antibody and an anti-Myc antibody, respectively.
Figure 10.
Porcine RNF122 performed K27-linked and K48-linked ubiquitination to MDA5. (a) HEK293T cells were co-transfected with Flag-MDA5 (1 μg) and Myc-RNF122 (0 μg, 0.5 μg, 1 μg, 2 μg), 12 h later, cells were infected with MG132 or not, 12 h later, the protein expression was tested by WB. (b,c) HEK293T cells were co-transfected with Myc-RNF122, Flag-MDA5 and HA-Ub (WT), HA-Ub (K48), HA-Ub (K2), HA-Ub (K33) or HA-Ub (K63). Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively. (d) Schematic of a mutant of Flag-MDA5. (e) HEK293T cells were co-transfected with Myc-RNF122, HA-Ub (WT) and Flag-MDA5, Flag-MDA5(K23R), Flag-MDA5 (K43R), Flag-MDA5 (K68R), Flag-MDA5 (K128R), Flag-MDA5 (K137R), Flag-MDA5 (K169R), Flag-MDA5 (K174R), Flag-MDA5 (K240R), Flag-MDA5 (K68RK137R) or not. Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively. (f) HEK293T cells were co-transfected with Myc-RNF122, HA-Ub (K27) and Flag-MDA5, Flag-MDA5 (K68RK137R) or not, 24 h after transfection, Flag Beads were used for CO-IP, and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively. (g) HEK293T cells were co-transfected with Myc-RNF122, HA-Ub (K48) and Flag-MDA5, Flag-MDA5 (K68RK137R) or not, 24 h after transfection, Flag Beads were used for CO-IP, and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively.
Figure 10.
Porcine RNF122 performed K27-linked and K48-linked ubiquitination to MDA5. (a) HEK293T cells were co-transfected with Flag-MDA5 (1 μg) and Myc-RNF122 (0 μg, 0.5 μg, 1 μg, 2 μg), 12 h later, cells were infected with MG132 or not, 12 h later, the protein expression was tested by WB. (b,c) HEK293T cells were co-transfected with Myc-RNF122, Flag-MDA5 and HA-Ub (WT), HA-Ub (K48), HA-Ub (K2), HA-Ub (K33) or HA-Ub (K63). Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively. (d) Schematic of a mutant of Flag-MDA5. (e) HEK293T cells were co-transfected with Myc-RNF122, HA-Ub (WT) and Flag-MDA5, Flag-MDA5(K23R), Flag-MDA5 (K43R), Flag-MDA5 (K68R), Flag-MDA5 (K128R), Flag-MDA5 (K137R), Flag-MDA5 (K169R), Flag-MDA5 (K174R), Flag-MDA5 (K240R), Flag-MDA5 (K68RK137R) or not. Flag Beads were used for CO-IP 24 h after transfection and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively. (f) HEK293T cells were co-transfected with Myc-RNF122, HA-Ub (K27) and Flag-MDA5, Flag-MDA5 (K68RK137R) or not, 24 h after transfection, Flag Beads were used for CO-IP, and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively. (g) HEK293T cells were co-transfected with Myc-RNF122, HA-Ub (K48) and Flag-MDA5, Flag-MDA5 (K68RK137R) or not, 24 h after transfection, Flag Beads were used for CO-IP, and further detected by Western blotting with an anti-HA, an anti-Flag antibody and an anti-Myc antibody, respectively.
Table 1.
The primers used for PCR amplification.
Table 1.
The primers used for PCR amplification.
Primer Name | Genbank Number | Sequence of Primer (5′-3′) |
---|
19T-RNF122-F | XM_013984096.2 | GCGCGTTCCTTGTCAGTTTT |
19T-RNF122-R | TTGCCACCCAACAGTCTTGT |
pCMV-RNF122-F | XM_013984096.2 | ccagtcgactctagaggatccATGCACCCATTTCAGTGGTGTA |
pCMV-RNF122-R | cagggatgccacccgggatccTCACACCAGTTCATCCAGTAGAATC |
pet-28a-RNF122-F | XM_013984096.2 | cagcaaatgggtcgcggatccATGCACCCATTTCAGTGGTGTA |
pet-28a-RNF122-R | acggagctcgaattcggatccTCACACCAGTTCATCCAGTAGAATC |
Myc-RNF122(TM)-F | XM_013984096.2 | ccagtcgactctagaggatccATGCACCCATTTCAGTGGTGTA |
Myc-RNF122(TM) -R | cagggatgccacccgggatccTCATTTAAGCACCACCTCTTTATATCC |
Myc-RNF122(RING)-F | XM_013984096.2 | ccagtcgactctagaggatccATGGGTGATGCCAAGAAGTTACA |
Myc-RNF122(RING) -R | cagggatgccacccgggatccTCACACCAGTTCATCCAGTAGAATC |
Flag-MDA5-F | NM_001100194.1 | ccagtcgactctagaggatccATGTCGTCGGATGGGTATTCC |
Flag-MDA5-R | cagggatgccacccgggatccTCAGTCCTCATCACTAGACAAACAATAT |
Flag-MDA5(N)-F | NM_001100194.1 | ccagtcgactctagaggatccATGTCGTCGGATGGGTATTCC |
Flag-MDA5(N)-R | cagggatgccacccgggatccTCAAGTCTCTTCATCTGAATCACTTCC |
Flag-MDA5(M)-F | NM_001100194.1 | ccagtcgactctagaggatccATGGTGGCTCAAAGAGCATCC |
Flag-MDA5(M)-R | cagggatgccacccgggatccTCAGGTGCTCTCATCAGCTCTG |
Flag-MDA5(C)-F | NM_001100194.1 | ccagtcgactctagaggatccATGTACGTCCTGGTTGCCCA |
Flag-MDA5(C)-R | cagggatgccacccgggatccTCAGTCCTCATCACTAGACAAACAATAT |
Flag-MDA5(K23R)-F | NM_001100194.1 | TGTTTCAGGGCCAGAGTGAGAAGGTACATT |
Flag-MDA5(K23R)-R | CTCACTCTGGCCCTGAAACACGAGATGAGA |
Flag-MDA5(K43R)-F | NM_001100194.1 | TTTCTGCCTGCAGAGGTGAGGGAGCAGATT |
Flag-MDA5(K43R)-R | CTCACCTCTGCAGGCAGAAAGGTCAAGTAG |
Flag-MDA5(K68R)-F | NM_001100194.1 | CTTCTGAACACTTTGGAGAGGGGGGTCTGG |
Flag-MDA5(K68R)-R | CTCTCCAAAGTGTTCAGAAGCAGTTCAGCT |
Flag-MDA5(K128R)-F | NM_001100194.1 | CAGCCTACAGTGGTGGACAGGCTTCTGGTT |
Flag-MDA5(K128R)-R | CTGTCCACCACTGTAGGCTGAAGAAGGTTC |
Flag-MDA5(K137R)-F | NM_001100194.1 | GTTACCGATGTCTTGGATAGATGTGTGGAG |
Flag-MDA5(K137R)-R | CTATCCAAGACATCGGTAACCAGAAGCTTG |
Flag-MDA5(K169R)-F | NM_001100194.1 | GGAGTAAGGGAGCTCCTGAGAAGGATTGTG |
Flag-MDA5(K169R)-R | CTCAGGAGCTCCCTTACTCCTGATTCATTT |
Flag-MDA5(K174R)-F | NM_001100194.1 | CTGAAAAGGATTGTGCAGAGAGAAAACTGG |
Flag-MDA5(K174R)-R | CTCTGCACAATCCTTTTCAGGAGCTCCCTT |
Flag-MDA5(K240R)-F | NM_001100194.1 | GACGTCTCGGACATAGAGAGAAGTTCACTG |
Flag-MDA5(K240R)-R | CTCTCTATGTCCGAGACGTCCAGACTTGGC |
Flag-nsp1α-F | JX317649.1 | caagcttgcggccgcgaattcaATGTCTGGGATACTTGATCGGTG |
Flag-nsp1α-R | cagggatgccacccgggatccTCAAGCACACTCAAAAGGGCA |
Flag-nsp1β-F | JX317649.1 | caagcttgcggccgcgaattcaATGGCTGACGTCTATGACATTGG |
Flag-nsp1β-R | cagggatgccacccgggatccTCAACCGTACCACTTATGACTGCC |
Flag-nsp4-F | JX317649.1 | aagcttgcggccgcgaattcaATGGGCGCTTTCAGAACTCA |
Flag-nsp4-R | cagggatgccacccgggatccTCATTCCAGTTCGGGTTTGG |
Flag-nsp5-F | JX317649.1 | caagcttgcggccgcgaattcaATGGGAGGCCTTTCCACAGT |
Flag-nsp5-R | cagggatgccacccgggatccTCACTCGGCAAAGTATCGCA |
Flag-nsp7-F | JX317649.1 | caagcttgcggccgcgaattcaATGTCGCTGACTGGTGCCC |
Flag-nsp7-R | cagggatgccacccgggatccTCATTCCCACTGAGCTCTTCTATTC |
Flag-nsp9-F | JX317649.1 | caagcttgcggccgcgaattcaATGTTTAAACTGCTAGCCGCCA |
Flag-nsp9-R | cagggatgccacccgggatccTCACTCATGATTGGACCTGAGTTT |
Flag-nsp10-F | JX317649.1 | caagcttgcggccgcgaattcaATGGGGAAGAAGTCCAGAATGTG |
Flag-nsp10-R | cagggatgccacccgggatccTCATTCCAGGTCTGCGCAA |
Flag-nsp11-F | JX317649.1 | caagcttgcggccgcgaattcaATGGGGTCGAGCTCCCCG |
Flag-nsp11-R | cagggatgccacccgggatccTCATTCAAGTTGAAAATAGGCCG |
Flag-N-F | JX317649.1 | caagcttgcggccgcgaattcaATGCCAAATAACAACGGCAAG |
Flag-N-R | cagggatgccacccgggatccTCATGCTGAGGGTGATGCTGT |
Flag-nsp4(K7R)-F | JX317649.1 | GGCGCTTTCAGAACTCAAAGGCCCTCACTG |
Flag-nsp4(K7R)-R | CTTTGAGTTCTGAAAGCGCCCATGAATTCG |
Flag-nsp4(K33R)-F | JX317649.1 | ACTATTGACGGGAAAATCAGGTGCGTGACT |
Flag-nsp4(K33R)-R | CTGATTTTCCCGTCAATAGTGAACACTCCG |
Flag-nsp4(K79R)-F | JX317649.1 | TGGCAAGGGGTTGCTCCCAGGGCCCAGTTC |
Flag-nsp4(K79R)-R | CTGGGAGCAACCCCTTGCCAATTCGGGCAA |
Flag-nsp4(K158R)-F | JX317649.1 | TGTAATGTGAAGCCCATCAGGCTGAGCGAG |
Flag-nsp4(K158R)-R | CTGATGGGCTTCACATTACAAAACTGGCCT |
Flag-nsp4(K170R)-F | JX317649.1 | GAATTCTTCGCTGGACCTAGGGTCCCGCTC |
Flag-nsp4(K170R)-R | CTAGGTCCAGCGAAGAATTCACTCAACTCG |
Table 2.
Primers used in small RNA interfering assay.
Table 2.
Primers used in small RNA interfering assay.
Primer Name | Primer Sequence (5′-3′) |
---|
negative control
| F:UUCUCCGAACGUGUCACGUTT |
R:ACGUGACACGUUCGGAGAATT |
siRNF122-1
| F: CCAUGCCACCCAUCAGUUUTT |
R:AAACUGAUGGGUGGCAUGGTT |
siRBF122-2
| F: GGACGAGCUAGGUGUGCUUTT |
R: AAGCACACCUAGCUCGUCCTT |
Table 3.
The primers used for Luciferase reporter gene.
Table 3.
The primers used for Luciferase reporter gene.
Primer Name | Genbank Number | Sequence of Primer (5′-3′) |
---|
pTP1-Luc-F
| NC_010457.5 | ggggtaccGGGATTGAACCCACATCCACA |
pTP1-Luc-R
| ccgctcgagACCACTGAAATGGGTGCATCA |
pTP2-Luc-F
| NC_010457.5 | ATGGATGGATGTTCCTGGCATCTCACCTCC |
pTP2-Luc-R
| ATCCATCCATGTGGATGTGGGTTCAATCCC |
pTP3-Luc-F
| NC_010457.5 | ATGGATGGATGGTCCCCAGGCTAGGGGTCC |
pTP3-Luc-R
| ATCCATCCATGTGGATGTGGGTTCAATCCC |
pTP4-Luc-F
| NC_010457.5 | ATGGATGGATGCCAGATCCTTAACCCACTG |
pTP4-Luc-R
| ATCCATCCATGTGGATGTGGGTTCAATCCC |
pTP5-Luc-F
| NC_010457.5 | ATGGATGGATTCTCTAGGTCCATCCCCCTT |
pTP5-Luc-R
| ATCCATCCATGTGGATGTGGGTTCAATCCC |
pTP6-Luc-F
| NC_010457.5 | ATGGATGGATTGCGTGCTGGCCGGTAAATA |
pTP6-Luc-R
| ATCCATCCATGTGGATGTGGGTTCAATCCC |
pTP7-Luc-F
| NC_010457.5 | ATGGATGGATGAGCTTCCCGGGGAGAGGGG |
pTP7-Luc-R
| ATCCATCCATGTGGATGTGGGTTCAATCCC |
pTP8-Luc-F
| NC_010457.5 | ATGGATGGATGTTCCTGGCATCTCACCTCC |
pTP8-Luc-R
| ATCCATCCATGTGGATGTGGGTTCAATCCC |
pTP9-Luc-F
| NC_010457.5 | ATGGATGGATCTGCCTTTCCTCGCCGTGGT |
pTP9-Luc-R
| ATCCATCCATGTGGATGTGGGTTCAATCCC |
pTP6-M1-Luc-F
| NC_010457.5 | CTCCCATTGGCTGGAGGGAAGGAGACGGGC |
pTP6-M1-Luc-R
| TTCCCTCCAGCCAATGGGAGCCTTCCTACG |
pTP6-M2-Luc-F
| NC_010457.5 | AAATAATACCCGGGGCCCAGCGGAAGGCTC |
pTP6-M2-Luc-R
| GCTGGGCCCCGGGTATTATTTACCGGCCAG |
pTP6-M3-Luc-F
| NC_010457.5 | CCGTAGGAAGGCTCCCATCAACTGGAGGGC |
pTP6-M3-Luc-R
| TTGATGGGAGCCTTCCTACGGGCCCCGGGT |
Table 4.
The primers used for qRT-PCR amplification.
Table 4.
The primers used for qRT-PCR amplification.
Primer Name | Genbank Number | Sequence of Primer (5′-3′) |
---|
RNF122-F
| XM_013984096.2 | ACATGGTCATCTTCGGCACA |
RNF122-R
| AGACTGCACAGGTCCCGTA |
PRRSV-N-F
| ABR37297.1 | CAGTCAATCAGCTGTGCCAAA |
PRRSV-N-R
| ATCTGACAGGGCACAAGTTCCA |
PRRSV-nsp2-F
| ABR37297.1 | CAGCCTTATGACCCCAACCAG |
PRRSV-nsp2-R
| TGGGCAAAGTCCCCTGTACCAA |
IFNβ-F
| NM_001003923 | GCAGTATTGATTATCCACGAGA |
IFNβ-R
| TCTGCCCATCAAGTTCCAC |
NF-κB -F
| X61498.1 | CCCAGCCATTTGCACACCTCAC |
NF-κB -R
| TTCAGAATTGCCCGACCAGTTTTT |
β-actin-F
| DQ452569.1 | GAATCCTGCGGCATCCACGA |
β-actin-R
| CTCGTCGTACTCCTGCTTGCT |
RB-F
| XM_013992198.2 | TCTCCTTTAAGATCCCCCAAGAA |
RB-R
| TTGAGGTTGCTTGTGCCTCT |
E2F1-F
| XM_021077692.1 | CGGCTTGAAGGATTGACCCA |
E2F1-R
| TCAGCATCCTCGGAAAGCAG |
E2F4-F
| XM_003126933.6 | AACGTGCTGGAAGGTATCGG |
E2F4-R
| CTTGTCCGCAATTTCCCGTG |
E2F6-F
| XM_005655271.3 | GTTGGATGTTCCTGCTCCCA |
E2F6-R
| CCGTCCGACACTTTACTGCT |
FOXC1-F
| XM_005665529.3 | GATGTTCGAGTCGCAGAGGAT |
FOXC1-R
| CAGAACTTGCTGCAGTCGTAG |
NFIC-F
| XM_021084124.1 | GGATGTATTCGTCCCCGCTC |
NFIC-R
| GTTGAACCAGGTGTAGGCGA |
HLTF-F
| XM_013991989.2 | TCGTGTTAGAGACCCAGCCT |
HLTF-R
| TCCAGGATCACTCTTAGCCAC |
Table 5.
Transcription factor binding sites of RNF122.
Table 5.
Transcription factor binding sites of RNF122.
Model ID | Model Name | Score | Relative Score | Start | End | Strand | Predicted Site Sequence |
---|
MA0032.1 | FOXC1 | 5.723 | 0.902187783821075 | 28 | 35 | 1 | GGCCCGTA |
MA0109.1 | HLTF | 6.251 | 0.901528565109457 | 43 | 52 | 1 | TCCCATTGGC |
MA0161.1 | NFIC | 8.520 | 0.960563817861358 | 48 | 53 | 1 | TTGGCT |
MA0024.2 | E2F1 | 14.481 | 0.985884368151812 | 56 | 66 | 1 | AGGGCGGGAGA |
MA0470.1 | E2F4 | 14.473 | 0.974005560828083 | 57 | 67 | 1 | GGGCGGGAGAC |
MA0471.1 | E2F6 | 14.993 | 0.9822249281057 | 57 | 67 | 1 | GGGCGGGAGAC |
MA0470.1 | E2F4 | 10.088 | 0.904564545543506 | 68 | 78 | 1 | GGGCGGGGGGC |
Table 6.
The type I interferon signaling pathway associated proteins in the RNF122 interaction proteins were identified by mass spectrometry.
Table 6.
The type I interferon signaling pathway associated proteins in the RNF122 interaction proteins were identified by mass spectrometry.
Description | Mass | Score |
---|
RING finger protein 122 (RNF122) | 18,204 | 1381 |
Transitional endoplasmic reticulum ATPase (VCP) | 89,950 | 93 |
Zinc finger CCCH-type antiviral protein 1 (ZC3HAV1) | 103,135 | 93 |
TNFAIP3-interacting protein 2 (TNIP2) | 49,240 | 33 |
Non-receptor tyrosine-protein kinase TYK2 (TYK2) | 135,389 | 31 |
Interferon-induced helicase C domain-containing protein 1 (MDA5) | 117,926 | 21 |
DNA-directed RNA polymerase III subunit RPC2 (POLR3B) | 129,242 | 20 |
E3 ubiquitin-protein ligase TRIM69 (TRIM69) | 58,351 | 17 |