pK205R targets the proximal element of IFN-I signaling pathway to assist African swine fever virus to escape host innate immunity at the early stage of infection

Figures

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  Fig 1. ASFV pK205R antagonizes IFN-I signaling. (A) HEK293T cells cultured in 24-well plates were transfected with ASFV K205R expression plasmid or empty vector (100 ng, 200 ng, or 400 ng), along with PRL-TK (25 ng) and ISRE-Luc (125 ng). At 24 hpt, cells were treated with 1,000 U/mL IFN-β for 8 h and analyzed using a dual-luciferase reporter assay. (B) HEK293T cells cultured in 24-well plates were transfected with ASFV K205R expression plasmid or empty vector (500 ng). After 24 h, cells were treated with 1,000 U/mL IFN-β for 8 h and analyzed through qPCR. (C) HEK293T cells were transfected with the pK205R-mCherry expression plasmid (200 ng, 400 ng, or 800 ng) or empty vector. At 16 hpt, cells were stimulated with 1,000 U/ml IFN-β and inoculated with VSV-GFP at an MOI of 0.1. At 24 hpt. Cells were cultured for an additional 24 h. GFP and mCherry signals were observed under a microscope (scale bar: 100 μm). *, P < 0.05; * *, P < 0.01; ***, P < 0.001.

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  Fig 2. pK205R inhibits the JAK/STAT signal transduction pathway. (A–C) HEK293T cells cultured in 12-well plates were transfected with pK205R expression plasmid (1 μg). At 24 hpt, cells were stimulated with or without IFN-β (1,000 U/mL) for 2 h. Expression of the indicated proteins was determined via western blotting. (D) pK205R (2.5 μg/ well) was transfected into HeLa cells in glass-bottom dishes. At 24 hpt, cells were stimulated with IFN-β (1,000 U/mL) for 2 h. Cells were stained with anti-STAT1/2 (red), and nuclei were stained with DAPI. Subcellular localization of the indicated proteins was analyzed by confocal microscopy (scale bar: 10 μm). (E) pK205R (2.5 μg/ well) was transfected into HEK293T cells in 6-well plates. At 24 hpt, the cells were stimulated with IFN-β (1,000 U/mL) for 2 h. Cytoplasmic and nuclear proteins were extracted from the cells using the NE-PER nuclear and cytoplasmic extraction kit. Levels of p-STAT1/2 in nuclear and cytoplasmic compartments was detected through western blotting. GAPDH and heat shock protein HDAC1 were used as cytoplasmic and nuclear markers, respectively.

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  Fig 3. pK205R interacts with both IFNAR1 and IFNAR2. (A, B) HEK293T cells were transfected with pK205R (5 μg) and either IFNAR1 or IFNAR2 (5 μg) in 100-mm petri dishes. At 24 hpt, cell lysates were subjected to Co-IP using HA and FLAG antibodies, followed by immunoblotting with FLAG and HA antibodies, respectively. (C) PAMs were infectted with ASFV-WT or ASFV-pK205R7PM in 100-mm dishes. Co-IP of cell lysates was performed using mouse pK205R antibodies at 3, 6, 12, and 24 hpi, followed by protein immunoblotting using rabbit pK205R, IFNAR1, and IFNAR2 antibodies. (D) pK205R (1 μg) and either IFNAR1 or IFNAR2 (1 μg) were co-transfected into HeLa cells in glass-bottom dishes. At 24 hpt, the cells were stained with anti-FLAG (green) and anti-HA (red) antibodies, and the nucleus was stained with DAPI (blue). Colocalization of the indicated proteins was analyzed via confocal microscopy (scale bar: 10 μm). For the fluorescence intensity analysis, red and green wavy lines indicate fluorescence of the corresponding color. The ordinate represents the gray value of the fluorescence channel, and the abscissa represents the relative position of the lines in the merged plot.

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  Fig 4. pK205R inhibits the binding of IFNAR1 with TYK2 and IFNAR2 with JAK1. (A, B) IFNAR1 (5 μg) and IFNAR2 (5 μg), or IFNAR1 (5 μg) and TYK2 (5 μg), or JAK1 (5 μg) and IFNAR2 (5 μg) were co-transfected into HEK293T cells with pK205R-EGFP (5 μg) or an empty vector in 100-mm dishes. Co-IP of cell lysates was performed using at 24 hpt using HA and FLAG antibodies, followed by immunoblotting with FLAG, HA, and EGFP antibodies. (C, D) IFNAR1 (5 μg) and TYK2 (5 μg) or JAK1 (5 μg) and IFNAR2 (5 μg) were co-transfected into HEK293T cells with varying doses of pK205R-EGFP (5 μg, 10 μg) or empty vector in 100 mm dishes. Co-IP of cell lysates was performed at 24 hpt using HA and FLAG antibodies, followed by immunoblotting using FLAG, HA, and EGFP antibodies.

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  Fig 5. Identification of key domains for pK205R interaction with IFNARs. (A) Schematic representation of the construction of truncated mutant plasmids for IFNAR1 and IFNAR2. (B, E) HEK293T cells in 100-mm dishes were co-transfected with pK205R (5 μg) and either IFNAR1/2 (5 μg), IFNAR1/2-ΔICD (5 μg), IFNAR1/2-ΔECD (5 μg), IFNAR1/2-ICD (5 μg), or IFNAR1/2-ECD (5 μg). At 24 hpt, cell lysates were subjected to Co-IP using HA and FLAG antibodies, followed by immunoblotting with FLAG and HA antibodies. (C, D, F, G) HeLa cells in glass-bottom dishes were transfected with pK205R (1 μg) and either IFNAR1/2-ΔICD (1 μg), IFNAR1/2-ΔECD (1 μg), IFNAR1/2-ICD (1 μg), or IFNAR1/2-ECD (1 μg), alone or in combination. At 24 hpt, cells were stained with anti-FLAG (green) and anti-HA (red) antibodies, and the nucleus was stained with DAPI (blue). Colocalization of the indicated proteins was analyzed by confocal microscopy (scale bar: 10 μm). For the fluorescence intensity analysis, red and green wavy lines indicate fluorescence of the corresponding color. The ordinate represents the gray value of the fluorescence channel, and the abscissa represents the relative position of the lines in the merged plot.

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  Fig 6. Identification of pK205R immunosuppressive functions of key domain structure. (A) Schematic representation of a deletion mutant constructed from the predicted pK205R functional domain. (B, C) HEK293T cells were co-transfected with pK205R (5 μg), PK205R-ΔCC (5 μg), and PK205R-ΔLCD (5 μg) along with IFNAR1 (5 μg) and IFNAR2 (5 μg) in 100-mm dishes. At 24 hpt, cell lysates were subjected to Co-IP using FLAG antibody, followed by immunoblotting with FLAG and HA. (D, E) HeLa cells were transfected with pK205R (1 μg), pK205R-ΔCC (1,000 ng), and pK205R-ΔLCD (1,000 ng) along with IFNAR1 (1 μg) and IFNAR2 (1 μg), alone or in combination. At 24 hpt, cells were stained with anti-FLAG (green) and anti-HA (red) antibodies, and the nucleus was stained with DAPI (blue). Colocalization of the indicated proteins was analyzed by confocal microscopy (scale bar: 10 μm). For the fluorescence intensity analysis, red and green wavy lines indicate fluorescence of the corresponding color. The ordinate represents the gray value of the fluorescence channel, and the abscissa represents the relative position of the lines in the merged plot. (F) HEK293T cells cultured in 24-well plates were co-transfected…

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  Fig 7. Identification of key amino acid sites mediating pK205R interaction with IFNAR1/2. (A, B) Docking model of pK205R with IFNAR1 and IFNAR2. IFNARs structure is shown in blue, TMD of IFNAR1 and IFNAR2 in red, pK205R structure in green, CCD in pink, and LCD domain in orange. (C, D) HEK293T cells in 100-mm culture dishes were co-transfected with pK205R (5 μg), pK205R7PM (5 μg), and either IFNAR1 (5 μg) or IFNAR2 (5 μg). Cell lysates were subjected to Co-IP using FLAG antibody, followed by immunoblotting with FLAG and HA antibodies. (E) HeLa cells in glass-bottom dishes were transfected with pK205R (1 μg), pK205R7PM (1 μg), and either IFNAR1 (1 μg) or IFNAR2 (1 μg), alone or in combination. At 24 hpt, cells were stained with anti-FLAG (green) and anti-HA (red) antibodies, and nuclei were stained with DAPI (blue). Colocalization of the indicated proteins was analyzed using confocal microscopy (scale bar: 10 μm). (F) HEK293T cells cultured in 24-well plates were co-transfected with pK205R (400 ng) or pK205R7PM (400 ng), along with PRL-TK (25 ng) and ISRE-Luc (125 ng), respectively. After 24 h, cells were treated with 1,000 U/mL IFN-β for 8 h and analyzed using a dual-luciferase reporter…

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  Fig 8. Construction of ASFV-pK205R7PM. (A) PAMs were infected with ASFV (MOI = 0.1), and cells were collected at the indicated time points post-infection. qPCR was used to detect the mRNA expression levels of K205R, CP204L, and B646L. (B) PAMs were infected with ASFV (MOI = 0.1), and cells were collected at the indicated time points post-infection. Cells were stained with anti-pK205R (green) and anti-p30 (red) antibodies, and nuclei were stained with DAPI (blue). Protein localization was analyzed by confocal microscopy (scale bar: 10 μm). (C) Schematic representation of ASFV-pK205R7PM constructed by homologous recombination and schematic representation of detection primers, the location of the mutation site is marked with a black triangle. (D) Green fluorescence signal of purified ASFV-pK205R7PM was observed under a fluorescence microscope. (E) Purity of recombinant strains was determined via PCR. Viral DNA obtained from the parental ASFV or ASFV-PK205R7PM mutant was amplified via PCR using the K205R and B646L primers described above, with B646L serving as an indicator of genomic input. (F) PAMs were infected with ASFV-WT (MOI = 0.01) or ASFV-pK205R7PM (MOI = 0.01), and the virus titers…

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  Fig 9. Effect of ASFV-WT and ASFV-pK205R7PM on IFN-I signaling. (A) PAMs were infected with ASFV-WT or ASFV-pK205R7PM (MOI = 0.1), and cells were harvested at the indicated time points for determination of ISG15, ISG54, and ISG56 mRNA levels. (B–D) PAMs were infected with ASFV-WT or ASFV-pK205R7PM (MOI = 0.1). At 24 hpi, cells were stimulated with IFN-β (1,000 U/mL) for 2 h. (B) Protein immunoblotting was performed using indicated antibodies. (C) Cells were stained with anti-pK205R or p30 (green) and anti-STAT1/2 (red) antibodies, and nuclei were stained with DAPI (blue). Protein localization was analyzed by confocal microscopy (scale bar: 10 μm). (D) p-STAT1/2 levels in nuclear and cytoplasmic compartments were detected using western blotting. GAPDH and heat shock protein HDAC1 were used as cytoplasmic and nuclear markers, respectively. (E) PAMs were infected with ASFV-WT or ASFV-pK205R7PM (MOI = 1) for 24 h and then treated with IFN-β (1,000 U/ml) for 8 h. mRNA levels of ISG15, ISG54, and ISG56 were determined using RT-qPCR. ns, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

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  Fig 10. ASFV pK205R antagonism of IFN-I signaling. ASFV pK205R targets IFNAR1 and IFNAR2, inhibiting the interaction between IFNAR1-TYK2 and IFNAR2-JAK1, thereby blocking downstream signaling pathways. Created with Biorender.com.