Loss of Ribosomal Protein RPL22 Restricts African Swine Fever Virus Replication by Inducing PERK-Dependent ER Stress

Figures

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  Figure 1.. Proximity Labeling using p54-TurboID Identifies Potential Host Interactors of ASFV p54. (A) Immunofluorescence assay of HEK-293 T cells transfected with V5-p54-TurboID, V5-TurboID-p54, and p54-ST. Cells were stained with anti-p54 antibodies. Scale bar, 500 µm. (B) Confocal microscopy of HEK-293 T cells co-expressing p54-ST (red) and V5-p54-TurboID (green). Enlarged view of boxed area is shown. Scale bar, 5 µm. (C) Optimization of biotinylation. HEK-293 T cells expressing V5-p54-TurboID were treated with various concentrations of biotin for different durations. Biotinylated proteins were detected using Streptavidin-HRP. GAPDH served as a loading control. (D) Western blot analysis of biotinylated proteins in HEK-293 T cells expressing V5-TurboID or V5-p54-TurboID following incubation with 500 µM biotin for 1 h. V5 tag blot confirms bait protein expression. (E) Schematic workflow of the TurboID-based proximity labeling experiment.

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  Figure 2.. Proteomic profiling and functional annotation of host proteins proximal to ASFV p54 identified using TurboID-based labeling. (A) Venn diagram showing the overlap of proteins identified in p54-TurboID and TurboID (control) samples. Numbers indicate unique protein counts. (B) Protein-protein interaction (PPI) network of the p54-proximal interactome. The network was constructed using the STRING database and visualized with Cytoscape. Each node represents a protein, and the edges represent interactions between them. (C) KEGG pathway enrichment analysis of the p54-proximal proteins. The Sankey diagram on the left shows the gene-term relationships, while the bubble plot on the right highlights the most significantly enriched pathways. The size of the bubbles corresponds to the number of enriched genes, and the color intensity represents the enrichment significance (-log10(P-value)). (D) Gene Ontology (GO) enrichment analysis of the p54-proximal candidate proteins, categorized by Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The bar chart displays the most significantly enriched GO terms, with bar length indicating significance (-log10(p-value)). Core…

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  Figure 3.. Validation of the Interaction Between p54 and Host Ribosomal Protein RPL22. (A) Co-immunoprecipitation (Co-IP) of exogenously expressed RPL22-Flag and p54-ST in HEK-293 T cells. Lysates were immunoprecipitated with anti-Flag or anti-ST antibodies. (B) Endogenous Co-IP in ASFV-infected (MOI = 1, 24 hpi) or mock-infected porcine alveolar macrophages (PAMs). Lysates were immunoprecipitated with anti-p54 or anti-RPL22 antibodies. IgG served as a negative control. (C) Confocal microscopy of HEK-293 T cells co-expressing RPL22-Flag (green) and p54-ST (red). Scale bar, 10 µm. (D) Bimolecular fluorescence complementation (BiFC) assay in HEK-293 T cells co-transfected with RPL22-nRFP and p54-cRFP. RFP signal indicates direct interaction. Scale bar, 10 µm.

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  Figure 4.. Knockout of RPL22 inhibits ASFV replication in LLC-PK1 cells. (A) Sanger sequencing of the targeted RPL22 locus in the RPL22-KO LLC-PK1-Cas9 cell line. The sgRNA target site and PAM sequence are indicated. (B) Western blot analysis of RPL22 expression in WT and RPL22-KO cells. GAPDH served as a loading control. (C) Fluorescence microscopy of WT and RPL22-KO cells infected with ASFV-GFP (MOI = 0.1) at 12, 24, and 48 hpi. Scale bar, 100 µm. (D) Quantification of ASFV genome copies in supernatants from (C) by qPCR. (E) Viral growth kinetics in WT and RPL22-KO cells infected with ASFV-GFP (MOI = 0.1). Viral titers were determined by TCID50 assay. Data in (D, E) are presented as mean ± SD (n = 3). Statistical significance was determined using Student's t-test. ns, not significant; *, p < 0.05; **, p < 0.01.

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  Figure 5.. Overexpression of RPL22 does not enhance ASFV replication in LLC-PK1 cells. (A) Western blot analysis of Flag-RPL22 expression in RPL22-overexpressing (RPL22-OE) and WT LLC-PK1 cells. GAPDH served as a loading control. (B) Fluorescence microscopy of WT and RPL22-OE cells infected with ASFV-GFP (MOI = 0.1) at 12, 24, and 48 hpi. Scale bar, 100 µm. (C) Quantification of ASFV genome copies in supernatants from (B) by qPCR. (D) Viral growth kinetics in WT and RPL22-OE cells infected with ASFV-GFP (MOI = 0.1). Data in (C, D) are shown as mean ± SD (n = 3). Statistical significance was determined by Student's t-test. ns, not significant.

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  Figure 6.. RPL22 Regulates ASFV Replication via Modulation of the PERK- eIF2α ER Stress Pathway(A) Relative mRNA levels of CHOP, BiP, and ATF4 in WT, ASFV-infected WT, and ASFV-infected RPL22-KO LLC-PK1-Cas9 cells (24 hpi), measured by qRT-PCR. (B) Western blot analysis of PERK pathway proteins in cells from (A). (C) qRT-PCR analysis of ER stress markers in WT, RPL22-KO, and RPL22-rescue cell lines. (D) Western blot analysis of PERK pathway proteins in cells from (C). (E) Western blot analysis of PERK pathway proteins in ASFV-infected WT and ASFV-infected RPL22-KO LLC-PK1-Cas9 cells (24 hpi), with or without treatment with the PERK inhibitor AMG-PERK44 (5 µM). (F) Fluorescence microscopy of WT, RPL22-KO, and AMG-PERK44-treated RPL22-KO cells infected with ASFV-GFP (MOI = 0.1, 24 hpi). Scale bar, 100 µm. (G) Quantification of ASFV genome copies in supernatants from (F) by qPCR. For all panels, GAPDH was used as a loading control. Data in (A, C, G) are shown as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

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  ← Figure 7.. Pharmacological modulation of general endoplasmic reticulum stress and the specific PERK pathway alters ASFV replication in vitro. (A) Fluorescence microscopy of LLC-PK1 cells pre-treated with Thapsigargin (Tg) or 4-phenylbutyric acid (4-PBA) for 6 h, then infected with ASFV-GFP (MOI = 0.1, 36 hpi). Scale bar; 10x-100 µm; 4x-250 µm. (B, C) Western blot analysis of BiP and p72 expression in LLC-PK1 (B) and PAM (C) cells treated as in (A). (D, E) qPCR quantification of ASFV genome copies in supernatants from LLC-PK1 (D) and PAM (E) cells with ASFV (MOI = 0.1, 36 hpi) after treatment with Tg or 4-PBA. (F) Fluorescence microscopy of LLC-PK1 cells pre-treated with CCT020312 or AMG-PERK44 for 6 h, then infected with ASFV-GFP (MOI = 0.1, 36 hpi). Scale bar, 100 µm. (G, H) Western blot analysis of p-PERK and p72 expression in LLC-PK1 (G) and PAM (H) cells treated as in (F). (I, J) Quantification of ASFV genome copies in supernatants from LLC-PK1 (I) and PAM (J) cells treated with CCT020312 or AMG-PERK44 and infected with ASFV (MOI = 0.1, 36 hpi). GAPDH was used as a loading control for all blots. Data in (D, E, I, J) are presented as mean ± SD (n = 3). Statistical significance was…

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  Figure 8.. A proposed model for the role of RPL22 as a key host factor in modulating the antiviral PERK signaling pathway to impact ASFV replication.