Highly pathogenic porcine reproductive and respiratory syndrome virus nonstructural protein 1 interacts with TRAF6 to activate the TAK1/p38/JNK/AP-1 signaling and induce IL-1β

Fig 1. PRRSV nsp1 upregulates IL-1β production. (A) PAMs were inoculated with HP-PRRSV at an MOI of 0.1. Total RNAs were extracted from cell lysates at 6, 12, 24, and 36 h post-inoculation, and qPCR was used to analyze IL-1β mRNA levels. The results were normalized to GAPDH. (B and C) PAMs were either mock-infected or infected with HP-PRRSV at MOIs of 0.01, 0.1, and 1 for 24 h, and total RNAs were extracted for qPCR analysis to analyze the mRNA levels of IL-1β and ORF7. (D) Supernatants were harvested at 6, 12, 24, and 36 h after HP-PRRSV infection (MOI = 0.1) to measure IL-1β production by ELISA. (E) PRRSV structural and nonstructural protein expression vectors (1 μg) were transfected into CRL-2843 cells. Total RNAs were extracted at 24 h, and qPCR was used to analyze IL-1β expression; the results were expressed as the fold induction over the vector control. (F) CRL-2843 cells were transfected with different doses of nsp1 expression vectors, and IL-1β mRNA was analyzed at 24 h by qPCR. (G) Supernatants were harvested at 6, 12, 24, and 36 h after nsp1 transfection (0.5 μg) to measure IL-1β production by ELISA. The data are representative of three independent experiments (means ± SEM).…

Fig 2. Nsp1 upregulates IL-1β expression through the JNK and p38 signaling pathways. (A) CRL-2843 cells were pretreated with inhibitors of p38 MAPK (SB203580, SB), JNK (SP600125, SP), ERK1/2 (HY12028, ERK1/2), PKC (PKC-IN-1, PKC), NF-κB (BAY11-7082, BAY), or DMSO (10 μM), and 1 h later, the cells were transfected with nsp1 (1 μg). After 24 h, IL-1β mRNA was analyzed by qPCR. (B and C) CRL-2843 cells were pretreated with p38 inhibitor (SB203580) and JNK inhibitor (SP600125) at different doses (2.5, 5, and 10 μM), and 1 h later, the cells were transfected with nsp1 (1 μg). After 24 h, the total RNAs were extracted for IL-1β mRNA analysis by qPCR. (D and F) CRL-2843 cells were transfected with p38 or JNK siRNA (60 nM), and NC was as the control. Cells were collected at 12, 24, and 48 h after transfection, and the mRNA level of p38 or JNK was detected by qPCR. (E and G) CRL-2843 cells were transfected with p38 or JNK siRNA (60 nM), and NC was as the control. At 12 h later, cells were transfected with nsp1 (1 μg). After 24 h, cells were collected, and the mRNA level of IL-1β was analysed by qPCR. (H) PAMs were transfected with nsp1 at different doses (0.5, 0.75, and 1 μg/mL), and the cells…

Fig 3. AP-1 is critical for PRRSV nsp1 to activate the IL-1β promoter. (A) Cloning and sequence analysis of the 2,112-bp porcine IL-1β gene 5′-flanking region. A schematic representation of the IL-1β promoter and the truncated promoter mutants. (B) The IL-1β promoter vector or pGL3 empty vector (0.5 μg) and nsp1 plasmid (0.5 μg) were transfected into HEK-293T cells, and the cells were then harvested to determine the luciferase activity at 24 h post-transfection. (C) Two AP-1 transcription factor-binding regions on the P4 promoter were deleted alternatively, and the AP-1-deleted luciferase reporter vectors were constructed: Δ1-luc, Δ2-luc, and ΔΔ-Luc. (D) Δ1-luc, Δ2-luc, or ΔΔ-Luc (0.5 μg) was co-transfected with nsp1 plasmid (0.5 μg) into HEK-293T cells. At 24 h post-transfection, cells were harvested to analyze the luciferase activity of the promoter. Differences were evaluated by Student's t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Fig 4. PRRSV nsp1-induced IL-1β expression is dependent on AP-1. (A) CRL-2843 cells were treated with AP-1 inhibitor SR-11302 at different doses (2.5, 5, and 10 μM) for 1 h, followed by transfection with the nsp1 plasmid (1 μg). Cells were collected at 24 h, and the mRNA level of IL-1β was analyzed by qPCR. (B) CRL-2843 cells were transfected with c-Jun siRNA (60 nM), with NC as the control. Cells were collected 12, 24, and 48 h after transfection, and the mRNA level of c-Jun was analyzed by qPCR. (C) CRL-2843 cells were transfected with c-Jun siRNA (60 nM), with NC as the control. The medium was changed at 12 h, followed by transfection with the nsp1 plasmid (1 μg). Cells were collected at 24 h, and the mRNA level of IL-1β was detected by qPCR. (D) Different doses of nsp1 plasmid (0.5, 0.75, and 1 μg/mL) were transfected into CRL-2843 cells, and the cells were collected at 24 h. The levels of p-c-Jun, c-Jun, Nsp1, and β-actin were analyzed by western blot. (E and F) CRL-2843 cells were transfected with JNK or P38 siRNA (60 nM), with NC as the control. The medium was changed at 12 h, followed by transfection with the nsp1 plasmid (1 μg). The cells were harvested at 24 h. Western…

Fig 5. PRRSV nsp1 activates JNK and P38 signaling pathways through TAK1 and TRAF6. (A–C) CRL-2843 cells were pretreated with the TAK1 inhibitor (5Z-7-Oxozeaenol), TRAF6 inhibitor (HY-120934), or IRAK inhibitor (HY-13329) at different doses, and 1 h later, the cells were transfected with nsp1 plasmid (1 μg). The total RNAs were extracted at 24 h post-transfection for IL-1β mRNA analysis by qPCR. (D and F) CRL-2843 cells were transfected with TAK1 or TRAF6 siRNA (60 nM), with NC as the control. Cells were collected 12, 24, and 48 h after transfection, and the mRNA level of TAK1 or TRAF6 was analyzed by qPCR. (E and G) CRL-2843 cells were transfected with TAK1 or TRAF6 siRNA (60 nM), with NC as the control. The medium was changed at 12 h, followed by transfection with the nsp1 plasmid (1 μg). Cells were collected at 24 h, and the mRNA level of IL-1β was analyzed by qPCR. (H) CRL-2843 cells were transfected with TRAF6 siRNA (30 or 60 nM), with NC as the control. The medium was changed at 12 h, followed by transfection with the nsp1 plasmid (1 μg). The cells were harvested at 24 h for western blot analysis. Differences were evaluated by Student's t-test. *, P < 0.05; **, P < 0.01; ns, not…

Fig 6. PRRSV nsp1 interacts with TRAF6. (A) HEK-293T cells were cotransfected with P4-luc promoter (0.5 μg) and Nsp1, Nsp1α, or Nsp1β plasmid (0.5 μg). The cells were harvested at 24 h post-transfection, and the luciferase activity was analyzed. (B) Nsp1, Nsp1α, or Nsp1β plasmid (1 μg) was transfected into CRL-2843 cells, and the IL-1β mRNA was analyzed by qPCR. (C) HEK-293T cells were cotransfected with Flag-Nsp1, Nsp1α, or Nsp1β (1 μg), and HA-TRAF6 plasmids (1 μg). The cells were harvested at 24 h later for Co-IP analysis. (D) HEK-293T cells were cotransfected with HA-Nsp1, Nsp1α, or Nsp1β (1 μg) and Flag-TRAF6 plasmids (1 μg). The cells were harvested at 24 h later for Co-IP analysis. (E) CRL-2843 cells were cotransfected with HA-Nsp1, Nsp1α, or Nsp1β plasmid (2 μg). The cells were harvested 24 h later for Co-IP analysis. (F) HEK-293T cells were cotransfected with Flag-Nsp1 (0.5 μg), Flag-TRAF6 (0.5 μg), and HA-Ub-k63 (1 μg). After 24 h, cells were treated with MG132 (20 μM) for 4 h, and then, the cells were harvested at 24 h later for Co-IP analysis. The ubiquitination levels of TRAF6 protein in IP and input were detected by western blot. Differences were evaluated by Student's…

Fig 7. Nsp1 R308 and R375 are the key amino acids for nsp1 to interact with TRAF6 and induce IL-1β production. (A) Model for the predicted target sites on nsp1 that might interact with TRAF6, including P134, K304, R308, P365, and R375. (B) CRL-2843 cells were transfected with nsp1 or each of the five mutants with the mutated amino acid at P134, K304, R308, P365, or R375 (1 μg). The IL-1β mRNA was quantified by qPCR. (C) HEK-293T cells were cotransfected with nsp1 or its five mutants and the P4 promoter. The cells were harvested at 24 h post-transfection, and the luciferase activity was analyzed. (D) Model for the mutant with mutations at both R308 and R375. (E) HEK-293T cells were cotransfected with wild-type nsp1, R308, R375, or R308/375 and the P4 promoter. The cells were harvested at 24 h post-transfection, and the luciferase activity was analyzed. (F) CRL-2843 cells were transfected with nsp1, R308, R375, or R308/375 (1 μg), and the IL-1β mRNA was quantified by qPCR. (G) HEK-293T cells were cotransfected with Flag-TRAF6 vector and nsp1 (R308A)-HA, nsp1 (R375A)-HA, nsp1 (R308/375A)-HA mutants, or nsp1-HA. The cells were harvested at 24 h post-transfection for Co-IP analysis.…

Fig 8. Nsp1 R308 and R375 are essential for HP-PRRSV replication and inflammation. (A) A mutant HP-PRRSV containing R308A and R375A mutations was constructed and named Mut. (B) PAMs were inoculated with the wild-type HP-PRRSV strain HV and the mutant virus PRRSV-R308/375A (Mut) at an MOI of 0.01, respectively. The cell supernatants were collected at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h post-inoculation, and the virus titer was detected. (C–F) Cells were harvested at the corresponding time points, and the mRNA levels of ORF7, IL-1β, IL-6, and TNFα were detected by qPCR. (G) PAMs were inoculated with HP-PRRSV strain HV and the mutant virus Mut (PRRSV-R308/375A) at an MOI of 0.01, respectively, and cell cytopathic effects were observed for 36 h post-infection. Differences were evaluated by Student's t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 9. Model for HP-PRRSV nsp1 to induce IL-1β production. After HP-PRRSV infection, the viral protein nsp1 induces the expression of IL-1β via interacting with TRAF6 to activate the TAK1/p38/JNK/AP-1 signaling pathway, in which nsp1 R308 and R375 are the key amino acids for nsp1 to induce IL-1β production.