Journal: Journal of Virology

Article Title: The S273R protein of African swine fever virus antagonizes the canonical NF- κ B signaling pathway by I κ B α

doi: 10.1128/jvi.02225-24

Figure Lengend Snippet: The ASFV pS273R inhibits the TNF- α - or IL-1 β -triggered NF- κ B signaling pathway. ( A ) The ASFV pS273R inhibits the TNF- α - or IL-1 β -triggered activation of the NF- κ B promoter in a dose-dependent manner. HEK293T cells were cotransfected with the different concentrations of the pS273R-expressing plasmid, pNF- κ B-Fluc, and pRL-TK. At 24 h post-transfection (hpt), the cells were treated with 20 ng/mL TNF- α or IL-1 β , followed by luciferase assay and western blotting analysis. ( B ) The ASFV pS273R inhibits the TNF- α - or IL-1 β -triggered transcription levels of proinflammatory cytokines. The HEK293T cells transfected with pFlag-S273R or the PK-15 cells stably expressing pS273R were treated with TNF- α or IL-1 β , and the total RNA was extracted for RT-qPCR assay. ( C ) The ASFV pS273R inhibits the TNF- α - or IL-1 β -triggered p65 phosphorylation. HEK293T cells transfected with pFlag-S273R or the PK-15 cells stably expressing pS273R were treated with TNF- α or IL-1 β for the indicated hpt. Western blotting analysis was performed using the indicated antibodies. The densitometric analysis of the protein expression levels was performed using ImageJ software. Ratio: target protein/ β -actin. The data shown were the means ± SDs from one representative experiment performed in triplicates ( A and B ). * P < 0.05; ** P < 0.01; *** P < 0.001; and ns, not significant ( P > 0.05) (unpaired Student’s t test).

Article Snippet: Rabbit MAb against the NF- κ B p65 (catalog no. 8242S), rabbit MAb against phospho-NF- κ B p65Ser536 (catalog no. 3033S), mouse MAb against I κ B α (catalog no. 4814S), and rabbit MAb against phospho-I κ B α Ser32 (catalog no. 2859S) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Plasmid Preparation, Transfection, Luciferase, Western Blot, Stable Transfection, Quantitative RT-PCR, Phospho-proteomics, Software

Journal: Journal of Virology

Article Title: The S273R protein of African swine fever virus antagonizes the canonical NF- κ B signaling pathway by I κ B α

doi: 10.1128/jvi.02225-24

Figure Lengend Snippet: The ASFV pS273R inhibits the NF- κ B signaling pathway independently of its protease activity. ( A ) Knockdown efficiency of the S273R -specific siRNAs. The S273R -specific siRNAs were transfected into the HEK293T cells overexpressing pS273R, followed by western blotting analysis using the indicated antibodies. ( B ) Knockdown of S273R induces elevated transcription levels of proinflammatory cytokines in the ASFV-infected PAMs. PAMs were transfected with the siRNAs, followed by ASFV infection. At 24 h postinfection (hpi), the cells were treated with 20 ng/mL TNF- α , and then, total RNA was extracted for RT-qPCR assay. ( C ) Knockdown of S273R elevates the p65 phosphorylation in the ASFV-infected PAMs. PAMs were transfected with the siRNAs for 12 h and infected with ASFV for 24 h. After treatment with TNF- α or left untreated at the indicated hpi, western blotting analysis was performed using the indicated antibodies. ( D ) Knockdown of S273R increases the TNF- α -induced cytokine production in the ASFV-infected PAMs. PAMs were transfected with the siRNAs infected with ASFV and then treated with TNF- α or left untreated. The IL-1 β expression level in the cell culture supernatants was detected using a commercial IL-1 β ELISA kit. ( E ) pS273R mutants inhibit the TNF- α -triggered activation of the NF- κ B promoter. HEK293T cells were cotransfected with the NF- κ B reporter, pRL-TK, and pFlag-S273R or pFlag-S273R mutants. At 24 h post-transfection (hpt), the cells were treated with 20 ng/mL TNF- α , followed by luciferase assay and western blotting analysis. ( F ) pS273R-3M inhibits the TNF- α -triggered phosphorylation of p65. PK-15 cells stably expressing pS273R or pS273R-3M were treated with TNF- α for the indicated minutes. Subsequently, the cells were lysed, followed by western blotting analysis using the indicated antibodies. ( G ) pS273R-3M inhibits the TNF- α -triggered transcription levels of proinflammatory cytokines in PK-15 cells. PK-15 cells stably expressing pS273R or pS273R-3M were treated with TNF- α , and the total RNA was extracted for RT-qPCR assay. The densitometric analysis of the protein expression levels was performed using the ImageJ software. Ratio: target protein/ β -actin ( C and F ). The data shown were the means ± SDs from one representative experiment performed in triplicates (B, D, E, and G). * P < 0.05; ** P < 0.01; *** P < 0.001; and ns, not significant ( P > 0.05) (unpaired Student’s t test).

Article Snippet: Rabbit MAb against the NF- κ B p65 (catalog no. 8242S), rabbit MAb against phospho-NF- κ B p65Ser536 (catalog no. 3033S), mouse MAb against I κ B α (catalog no. 4814S), and rabbit MAb against phospho-I κ B α Ser32 (catalog no. 2859S) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Activity Assay, Knockdown, Transfection, Western Blot, Infection, Quantitative RT-PCR, Phospho-proteomics, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Luciferase, Stable Transfection, Software

Journal: Journal of Virology

Article Title: The S273R protein of African swine fever virus antagonizes the canonical NF- κ B signaling pathway by I κ B α

doi: 10.1128/jvi.02225-24

Figure Lengend Snippet: The ASFV pS273R is associated with the NF- κ B complex. ( A ) ASFV pS273R functioned at or upstream of p65. HEK293T cells were cotransfected with pNF- κ B-Fluc, pRL-TK, and the indicated expression plasmids, followed by luciferase assay. ( B and C ) ASFV pS273R interacts with I κ B α , p65, or p50. HEK293T cells were cotransfected with the plasmids expressing the HA-TAK1, -TAB1, -IKK β , -I κ B α , -p65, or -p50, and pFlag-S273R, and then lysed for co-IP using anti-HA or anti-Flag MAb, followed by western blotting analysis using the indicated antibodies. ( D ) The ASFV pS273R binds to I κ B α , p65, and p50 in vitro . The recombinant GST-tagged pS273R was incubated with the HA-tagged I κ B α , p65, or p50, followed by western blotting using the indicated antibodies. ( E ) The ASFV pS273R interacts with the endogenous I κ B α , p65, or p50. PAMs were infected with ASFV-WT. At 24 hpi, the cells were lysed for co-IP using anti-pS273R PAb, followed by western blotting analysis using the indicated antibodies. ( F ) The ASFV pS273R is colocalized with I κ B α , p65, or p50 mainly in the cytoplasm. PAMs were infected with ASFV-WT. At 24 hpi, the cells were fixed with 4% paraformaldehyde. I κ B α , p65, p50, and pS273R were immunoblotted using anti-I κ B α , -p65, -p50, and -pS273R antibodies, respectively. The nuclei were stained with DAPI and subjected to confocal microscopy analysis. The colocalization of pS273R and the molecules tested was analyzed using the Coloc2 tool of ImageJ and shown as Pearson’s correlation coefficients. Scale bar = 10 µm.

Article Snippet: Rabbit MAb against the NF- κ B p65 (catalog no. 8242S), rabbit MAb against phospho-NF- κ B p65Ser536 (catalog no. 3033S), mouse MAb against I κ B α (catalog no. 4814S), and rabbit MAb against phospho-I κ B α Ser32 (catalog no. 2859S) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Expressing, Luciferase, Co-Immunoprecipitation Assay, Western Blot, In Vitro, Recombinant, Incubation, Infection, Staining, Confocal Microscopy

Journal: Journal of Virology

Article Title: The S273R protein of African swine fever virus antagonizes the canonical NF- κ B signaling pathway by I κ B α

doi: 10.1128/jvi.02225-24

Figure Lengend Snippet: The ASFV pS273R does not affect the transcription, ubiquitination, and SUMOylation of I κ B α . ( A ) The diagram of the dynamic regulation of I κ B α by IKK β . ( B ) The ASFV pS273R does not impair the transcription of I κ B α . HEK293T cells were transfected with pFlag-S273R or pRK (Vec) and then treated with 20 ng/mL TNF- α or IL-1 β at various times. The total RNA was extracted for RT-qPCR assay. ( C ) The ASFV pS273R significantly suppresses the degradation of I κ B α induced by TNF- α . HEK293T cells were transfected with pFlag-S273R or Vec. The cells were then treated with TNF- α at the indicated hpt, followed by western blotting analysis using the indicated antibodies. The densitometric analysis of the protein expression levels was performed using ImageJ software. Ratio: I κ B α / β -actin. ( D ) The ASFV pS273R does not affect the polyubiquitination of I κ B α . HEK293T cells were cotransfected with the HA-Ub-WT-, Flag-I κ B α -, and Myc-pS273R-expressing plasmids, followed by co-IP and western blotting analysis using the indicated antibodies. ( E ) The ASFV pS273R does not affect the interaction between ubiquitinated I κ B α and p65-p50. HEK293T cells were cotransfected with the plasmids expressing HA-Ub, Flag-I κ B α , and Myc-pS273R and treated with MG132 , followed by co-IP and western blotting analysis using the indicated antibodies. ( F ) The ASFV pS273R does not affect the SUMOylation of I κ B α . HEK293T cells were cotransfected with the HA-SUMO1-, Flag-I κ B α -, Myc-UBC9-, and Myc-pS273R-expressing plasmids, followed by co-IP and western blotting using the indicated antibodies. The data shown were the means ± SDs from one representative experiment performed in triplicates ( B ). * P < 0.05; ** P < 0.01; *** P < 0.001; and ns, not significant ( P > 0.05) (unpaired Student’s t test).

Article Snippet: Rabbit MAb against the NF- κ B p65 (catalog no. 8242S), rabbit MAb against phospho-NF- κ B p65Ser536 (catalog no. 3033S), mouse MAb against I κ B α (catalog no. 4814S), and rabbit MAb against phospho-I κ B α Ser32 (catalog no. 2859S) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Ubiquitin Proteomics, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Software, Co-Immunoprecipitation Assay

Journal: Journal of Virology

Article Title: The S273R protein of African swine fever virus antagonizes the canonical NF- κ B signaling pathway by I κ B α

doi: 10.1128/jvi.02225-24

Figure Lengend Snippet: The ASFV pS273R inhibits the nuclear translocation of p65. ( A ) ASFV pS273R reduces the distribution of p65 in the nucleus. HEK293T cells were transfected with pFlag-S273R or pRK (Vec). The cells were then treated with 20 ng/mL TNF- α at the indicated hpt. The cells were then concentrated and lysed, followed by subcellular fractionation and western analysis blotting using the indicated antibodies. ( B ) The ASFV pS273R inhibits the nuclear translocation of p65. The stable cell lines overexpressing pS273R were treated with 20 ng/mL TNF- α or IL-1 β and then fixed for immunoblotting followed by confocal microscopy analysis. The colocalization of p65 and DAPI was analyzed using the Coloc2 tool of ImageJ/FIJI and was shown as Pearson’s correlation coefficients. Scale bar = 10 µm. ( C ) Knockdown of pS273R attenuates the ability of ASFV to antagonize the TNF- α -induced nuclear translocation of p65. PAMs were transfected with the siRNAs for 12 h and then infected with ASFV for 24 h, followed by treatment with TNF- α or left untreated. The cells were then concentrated and lysed, followed by subcellular fractionation and subjected to western blotting analysis using the indicated antibodies.

Article Snippet: Rabbit MAb against the NF- κ B p65 (catalog no. 8242S), rabbit MAb against phospho-NF- κ B p65Ser536 (catalog no. 3033S), mouse MAb against I κ B α (catalog no. 4814S), and rabbit MAb against phospho-I κ B α Ser32 (catalog no. 2859S) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Translocation Assay, Transfection, Fractionation, Western Blot, Stable Transfection, Confocal Microscopy, Knockdown, Infection

Journal: Journal of Virology

Article Title: The S273R protein of African swine fever virus antagonizes the canonical NF- κ B signaling pathway by I κ B α

doi: 10.1128/jvi.02225-24

Figure Lengend Snippet: The amino acids 83–273 of the ASFV pS273R are essential for suppressing the NF- κ B signaling pathway. ( A ) Schematic illustration of the truncated pS273R mutants. ( B ) The different domains of pS273R interact with the NF- κ B complex. HEK293T cells were cotransfected with the plasmids expressing the HA-I κ B α , -p65, or -p50, and pFlag-S273R or the plasmids expressing the truncated pS273R mutants and then lysed for co-IP assay using anti-Flag MAb, followed by western blotting analysis using the indicated antibodies. ( C ) The aa 83–273 of pS273R are sufficient to inhibit the TNF- α -triggered activation of the NF- κ B promoter. HEK293T cells were cotransfected with the plasmids expressing the NF- κ B reporter, pRL-TK, and pFlag-S273R or the plasmids expressing the truncated versions, followed by luciferase assay. ( D ) The aa 83–273 of pS273R remarkably impaired the TNF- α -triggered phosphorylation of p65. HEK293T cells were transfected with pFlag-S273R or the plasmids expressing the truncated versions of pS273R and treated with 20 ng/mL TNF- α for the indicated time, subjected to Western blotting analysis using the indicated antibodies. The densitometric analysis of the protein expression levels was performed using ImageJ software. Ratio: target protein/ β -actin. ( E ) The aa 83–273 of pS273R reduce the TNF- α -triggered transcription levels of the IL-6 , IL-8 , and TNF-α genes. HEK293T cells were transfected with pFlag-S273R or the plasmids expressing the truncated versions and treated with 20 ng/mL TNF- α , and then the total RNA was isolated for the RT-qPCR assay. The data shown were the means ± SDs from one representative experiment performed in triplicates ( C and E ). ***, P < 0.001 (unpaired Student’s t test).

Article Snippet: Rabbit MAb against the NF- κ B p65 (catalog no. 8242S), rabbit MAb against phospho-NF- κ B p65Ser536 (catalog no. 3033S), mouse MAb against I κ B α (catalog no. 4814S), and rabbit MAb against phospho-I κ B α Ser32 (catalog no. 2859S) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot, Activation Assay, Luciferase, Phospho-proteomics, Transfection, Software, Isolation, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: The S273R protein of African swine fever virus antagonizes the canonical NF- κ B signaling pathway by I κ B α

doi: 10.1128/jvi.02225-24

Figure Lengend Snippet: Schematic diagram of the mechanism by which the ASFV pS273R suppresses host inflammatory responses. The ASFV pS273R inhibits the TNF- α - or IL-1 β -triggered inflammatory responses by targeting I κ B α . pS273R inhibits the degradation of I κ B α by disturbing the association of I κ B α with the proteasome, leading to the reduced phosphorylation and nuclear translocation of p65 and subsequently the lower expression levels of proinflammatory cytokines.

Article Snippet: Rabbit MAb against the NF- κ B p65 (catalog no. 8242S), rabbit MAb against phospho-NF- κ B p65Ser536 (catalog no. 3033S), mouse MAb against I κ B α (catalog no. 4814S), and rabbit MAb against phospho-I κ B α Ser32 (catalog no. 2859S) were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Phospho-proteomics, Translocation Assay, Expressing