Fig 1.
The KP177R gene is not essential for ASFV replication in porcine alveolar macrophages (PAMs).
(A) The recombinant virus ASFV-ΔKP177R was analyzed using direct fluorescence analysis. (B) Gene sequencing analysis of the viral genome. (C) PAMs were infected with ASFV-WT or ASFV-ΔKP177R (MOI = 0.1) for the indicated hours, and western blotting analysis was performed. (D) ASFV-WT-infected PAMs were fixed and immunolabeled with a mouse antibody against p22 followed by an anti-mouse antibody conjugated to 10-nm-diameter gold particles. The subviral localization of p22 (red arrows) was observed by immunoelectron microscopy. The gold particles present on intracellular viruses were associated with the inner envelope (green) beneath the outer protein capsid (red). (E) The morphology of the mature virions (red arrows) was observed in the ASFV-ΔKP177R- or ASFV-WT-infected PAMs (MOI = 3) using transmission electron microscopy. (F) PAMs were infected with ASFV-ΔKP177R or ASFV-WT (MOI = 0.1). Hemadsorption property of the viruses was examined using microscopy. (G) PAMs were seeded into 24-well plates and infected with ASFV-ΔKP177R or ASFV-WT (MOI = 0.01). At the indicated time points, the genome copies and viral titers were determined using RT-qPCR and hemadsorption assays, respectively.
Fig 2.
The KP177R-deleted ASFV mutant significantly promotes the activation of the JAK-STAT signaling pathway in PAMs.
(A) The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed in the ASFV-ΔKP177R- vs ASFV-WT-infected PAMs (MOI = 3). (B) Heat map of the ISGs in the ASFV-ΔKP177R- vs ASFV-WT-infected PAMs (MOI = 3). (C) PAMs were infected with ASFV-WT or ASFV-ΔKP177R (MOI = 3). At 6 hpi, the cells were treated with 400 ng/mL IFN-β for 6 h. The total RNA was extracted using TRIzol reagent. Subsequently, the mRNA transcription of indicated genes was examined by RT-qPCR. (D) PAMs were infected with ASFV-WT or ASFV-ΔKP177R (MOI = 3). At 6 hpi, the cells were treated with 20 ng/mL TNF-α for 6 h, and mRNA transcription of indicated genes was determined as described above. (E) PAMs were infected with ASFV-WT or ASFV-ΔKP177R (MOI = 3). The PAMs were either treated with IFN-β at 12 hpi, and western blotting analysis was performed using the indicated antibodies.
Fig 3.
The ASFV p22 inhibits the IFN-β-triggered activation of the JAK-STAT signaling pathway.
(A) HEK293T cells were cotransfected with different amounts of the Flag-p22-expressing plasmid and pSTAT1-Fluc. At 24 hours posttransfection (hpt), the cells were treated with IFN-β, followed by luciferase assay and western blotting analysis. (B) HEK293T cells were cotransfected with the different amounts of the Flag-p22-expressing plasmid, pNF-κB-Fluc, and pRL-TK. At 24 hpt, the cells were treated with 20 ng/mL TNF-α and subjected to luciferase assay and western blotting analysis. (C) HEK293T cells transfected with pFlag-KP177R or the IBRS-2 cells stably expressing p22 were treated with 200 ng/mL IFN-β for the indicated hours. Western blotting analysis was performed using the indicated antibodies. (D) HEK293T cells transfected with pFlag-KP177R or IBRS-2 cells stably expressing p22 were treated with IFN-β. The cells were subjected to isolation of cellular total RNAs followed by quantification of transcription level of indicated genes by RT-qPCR. (E) HEK293T cells transfected with pFlag-KP177R or the IBRS-2 cells stably expressing p22 were treated with 20 ng/mL TNF-α, and the total RNA was extracted and analyzed using RT-qPCR as described above. The data were shown as the means ± SDs from one representative experiment performed in triplicates. * P < 0.05; ** P < 0.01; *** P < 0.001; and ns, not significant (P > 0.05) (unpaired Student’s t test).
Fig 4.
The ASFV p22 targets IFNAR1 and promotes its degradation.
(A) HEK293T cells were cotransfected with the plasmids expressing HA-IFNAR1 or HA-IFNAR2, and Flag-p22 or the empty vector pRK (Vec) and then lysed and analyzed using co-IP with the anti-Flag MAb, followed by western blotting analysis using the indicated antibodies. (B) PAMs were infected with ASFV-WT or ASFV-ΔP (MOI = 3). The cells were lysed for co-IP using the anti-p22 MAb at 24 hpi, followed by western blotting analysis using the indicated antibodies. (C) IBRS-2 cells were cotransfected with the plasmids expressing Myc-IFNAR1 and Flag-p22 and then fixed with 4% paraformaldehyde. IFNAR1 and p22 were immunoblotted using anti-Myc and anti-Flag antibodies, respectively. The nuclei were stained with DAPI and subjected to confocal microscopy. The colocalization of p22 and IFNAR1 was analyzed using the Coloc2 tool in the ImageJ software (National Institutes of Health, Bethesda, MD, USA), and they are represented as Pearson’s R value (R). Scale bar = 10 μm. (D) HEK293T cells were cotransfected with the HA-β-actin-, Flag-p22-, and HA-IFNAR1- or HA-IFNAR2-expressing plasmid, followed by western blotting analysis using the indicated antibodies at 24 hpt. (E) HEK293T cells were transfected with different amounts of the Flag-p22-expressing plasmid, followed by western blotting analysis using the indicated antibodies at 24 hpt. (F) PAMs were infected with ASFV-WT or ASFV-ΔKP177R (MOI = 3). At 18 and 24 hpi, the cells were lysed to perform western blotting analysis using the indicated antibodies.
Fig 5.
The ASFV p22 degrades IFNAR1 via autophagy.
(A) HEK293T cells were cotransfected with the plasmids expressing HA-IFNAR1, HA-β-actin, and Flag-p22 or pRK (Vec). Then, the cells were treated with DMSO, MG132, or CQ at 24 hpt, followed by western blotting analysis using the indicated antibodies. (B) HEK293T cells were transfected with the plasmids pFlag-KP177R or pRK (Vec). Then, the cells were treated with DMSO, SBI, 3-MA, or CQ at 24 hpt, followed by western blotting analysis using the indicated antibodies. (C) HEK293T cells were transfected with the different amounts of the Flag-p22-expressing plasmid with pHA-IFNAR1 and pHA-β-actin, followed by western blotting analysis using the indicated antibodies at 24 hpt. (D) IBRS-2 cells were cotransfected with the plasmids expressing EGFP-LC3, Myc-IFNAR1, and Flag-p22, and then fixed with 4% paraformaldehyde. IFNAR1 and p22 were immunoblotted using anti-Myc and -Flag antibodies, respectively. The nuclei were stained with DAPI and analyzed using confocal microscopy. The colocalization of LC3 and IFNAR1 was analyzed using the Coloc2 tool in ImageJ and are shown as Pearson’s R value (R). Scale bar = 10 μm. (E) PAMs were infected with ASFV-WT or ASFV-ΔKP177R for the indicated duration (MOI = 3), followed by western blotting analysis using the indicated antibodies. (F) The ATG5-knockout HeLa cells or the WT HeLa cells were transfected with the different amounts of the Flag-p22-expressing plasmid with pHA-IFNAR1 and pHA-β-actin, followed by western blotting analysis using the indicated antibodies at 24 hpt. The densitometric analysis of the protein expression levels was performed using the ImageJ software.
Fig 6.
TAX1BP1 is involved in the p22-induced degradation of IFNAR1.
(A) HEK293T cells were cotransfected with the plasmids expressing HA-NBR1, -CALCOCO2, -TAX1BP1, -p62, -OPTN, -NIX, or -TOLLIP, and Flag-p22, and then lysed for co-IP using the anti-Flag MAb, followed by western blotting analysis using the indicated antibodies. (B) HEK293T cells were cotransfected with the plasmids expressing Flag-p22 and HA-TAX1BP1, and then lysed for Co-IP using anti-Flag or anti-HA MAb, followed by western blotting analysis using the indicated antibodies. (C) HEK293T cells were transfected with pHA-TAX1BP1, harvested and lysed at 24 hpt, and the supernatants were incubated with the prokaryotically expressed GST-p22 protein bound to the GST beads. The formed complexes were analyzed using western blotting and the indicated antibodies. (D) IBRS-2 cells were cotransfected with the plasmids expressing HA-TAX1BP1 and Flag-p22, and then fixed with 4% paraformaldehyde. TAX1BP1 and p22 were immunoblotted using anti-HA and anti-Flag antibodies, respectively. The nuclei were stained with DAPI and subjected to confocal microscopy. The colocalization of p22 and TAX1BP1 was analyzed using the Coloc2 tool in ImageJ, and it is shown as Pearson’s R value (R). Scale bar = 10 μm. (E) PAMs were infected with ASFV-WT or ASFV-ΔKP177R (MOI = 3). At 24 hpi, the cells were lysed for co-IP using anti-p22 MAb, followed by western blotting analysis using the indicated antibodies. (F) HEK293T cells were transfected with different amounts of the HA-TAX1BP1-expressing plasmid with pFlag-KP177R, followed by western blotting analysis using the indicated antibodies at 24 hpt. (G) The TAX1BP1-knockout or WT HEK293T cells were transfected with different amounts of the Flag-p22-expressing plasmid or pRK (Vec), followed by western blotting analysis using the indicated antibodies at 24 hpt. The densitometric analysis of the protein expression levels was performed using the ImageJ software.
Fig 7.
p22 enhances the TAX1BP1-mediated degradation of IFNAR1.
(A) HEK293T cells were cotransfected with the plasmids expressing the Myc-IFNAR1, HA-β-actin, HA-TAX1BP1, and Flag-p22, followed by western blotting analysis using the indicated antibodies. (B) HEK293T cells were cotransfected with the plasmids expressing Myc-IFNAR1, HA-β-actin, and HA-TAX1BP1. Then, the cells were treated with DMSO, SBI, 3-MA, or CQ at 24 hpt, followed by western blotting analysis using the indicated antibodies. (C) HEK293T cells were transfected with different amounts of the TAX1BP1- or TAX1BP1-ΔUBD-expressing plasmids with pMyc-IFNAR1 and pHA-β-actin, followed by western blotting analysis using the indicated antibodies at 24 hpt. (D) HEK293T cells were cotransfected with the plasmids expressing Myc-IFNAR1, HA-β-actin, Flag-p22, and HA-TAX1BP1 or HA-TAX1BP1-ΔUBD, followed by western blotting analysis using the indicated antibodies. (E) HEK293T cells were cotransfected with the plasmids expressing Myc-IFNAR1, HA-Ub, and Flag-p22, and then lysed for co-IP using anti-Myc MAb, followed by western blotting analysis using the indicated antibodies. (F) HEK293T cells were transfected with different amounts of the p22-expressing plasmid with pMyc-IFNAR1 and pHA-TAX1BP1, and then lysed for co-IP using anti-HA MAb at 24 hpt, followed by western blotting analysis using the indicated antibodies.
Fig 8.
The transmembrane domain of the ASFV p22 is essential for inhibiting the activation of the JAK-STAT signaling pathway.
(A) Schematic illustration of the domain structures of p22, IFNAR1, and TAX1BP1, and the construction strategy of their truncation mutants. (B) HEK293T cells were cotransfected with plasmids pSTAT1-Fluc and pFlag-KP177R or plasmids expressing the truncated versions at 24 hpt and luciferase assay was conducted. (C) IBRS-2 cells were transfected with pFlag-KP177R or pFlag-KP177R-ΔECD, and treated with 200 ng/mL IFN-β, followed by total RNA extraction and the subsequent RT-qPCR analysis. (D) IBRS-2 cells were transfected with pFlag-KP177R or pFlag-KP177R-ΔECD and treated with 200 ng/mL IFN-β for the indicated hours and subjected to western blotting analysis using the indicated antibodies. (E) HEK293T cells were cotransfected with the plasmids expressing Myc-IFNAR1, HA-β-actin, and Flag-p22 or the truncated versions of p22 and subjected to western blotting analysis using the indicated antibodies. The densitometric analysis of the protein expression levels was performed using the ImageJ software.
Fig 9.
Schematic diagram of the mechanism by which the ASFV p22 suppresses cellular IFN responses.
The selective autophagy receptor TAX1BP1 interacts with and degrades IFNAR1 via autophagy. The transmembrane region of the ASFV p22 enhances the binding of TAX1BP1 to IFNAR1, thereby promoting the degradation of IFNAR1. This leads to a decrease in the transcriptional levels of antiviral genes, ultimately inhibiting the host IFN responses. Created with BioRender.com.