Journal: Journal of Virology

Article Title: 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

doi: 10.1128/jvi.00428-26

Figure Lengend Snippet: 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 significant.

Article Snippet: Membranes were blocked with 5% skim milk in PBS for 1 h at room temperature, followed by incubation overnight at 4°C with antibodies against p-c-Jun, c-Jun, p-p38, p38, p-JNK, JNK, p-TAK1, TAK1, and TRAF6 (MCE, USA).

Techniques: Protein-Protein interactions, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Journal of Virology

Article Title: 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

doi: 10.1128/jvi.00428-26

Figure Lengend Snippet: 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 t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Membranes were blocked with 5% skim milk in PBS for 1 h at room temperature, followed by incubation overnight at 4°C with antibodies against p-c-Jun, c-Jun, p-p38, p38, p-JNK, JNK, p-TAK1, TAK1, and TRAF6 (MCE, USA).

Techniques: Plasmid Preparation, Transfection, Luciferase, Activity Assay, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Western Blot

Journal: Journal of Virology

Article Title: 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

doi: 10.1128/jvi.00428-26

Figure Lengend Snippet: 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. Differences were evaluated by Student's t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: Membranes were blocked with 5% skim milk in PBS for 1 h at room temperature, followed by incubation overnight at 4°C with antibodies against p-c-Jun, c-Jun, p-p38, p38, p-JNK, JNK, p-TAK1, TAK1, and TRAF6 (MCE, USA).

Techniques: Transfection, Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Journal of Virology

Article Title: 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

doi: 10.1128/jvi.00428-26

Figure Lengend Snippet: 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.

Article Snippet: Membranes were blocked with 5% skim milk in PBS for 1 h at room temperature, followed by incubation overnight at 4°C with antibodies against p-c-Jun, c-Jun, p-p38, p38, p-JNK, JNK, p-TAK1, TAK1, and TRAF6 (MCE, USA).

Techniques: Infection, Expressing