Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

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  Figure 1.. RNF149 expression is upregulated by EMCV. (A–D) A549 and HEK293T cells were infected with EMCV at 0.1 MOI were harvested post infection 24 hours, RT-qPCR was performed to detect the mRNA levels of RNF6, RNF19B, RNF24, RNF149, RNF157, RNF182, RNF186, RNF123, RNF14, RNF43, RNF152, RNF207, RNF208, and RNF224 mRNA. (E-F) A549 and HEK293T cells were infected with EMCV at 0.1 MOI were harvested post infection 0 h, 6 h, 9 h, 12 h, and 24 h, protein level measured using anti-RNF149 and anti-EMCV-VP1 antibodies. β-actin was used as a loading control. The results are presented as means ± sd. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

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  Figure 2.. RNF149 promotes EMCV replication. (A) HEK293T cells were transfected with negative control siRNA (siRNA-ctrl) or siRNA-1#, −2#, −3#, and −4# against RNF149. Western blotting was used to analysis the protein expression level with anti-RNF149. β-actin was used as a loading control. (B) A549 cells were transfected with negative control siRNA-ctrl, siRNA-4# for 48 h and then infected with EMCV at 0.1 MOI were harvested at 24 hpi, RT-qPCR detect EMCV mRNA. Virus titers were analyzed by TCID50 assay. (C) HEK293T cells were transfected with negative control siRNA-ctrl, siRNA-4# for 48 h and then infected with EMCV at 0.1 MOI were harvested at 24 hpi, RT-qPCR detect EMCV mRNA. Virus titers were analyzed by TCID50 assay. (D) HEK293T cells were transfected with negative control siRNA-ctrl, siRNA-4# for 48 h and then the cells were infected with VSV-GFP at 0.01 MOI for 18 h, and the cells were observed with fluorescence microscopy. Scale bars: 300 µm. (E) VSV mRNA level was detected by RT-qPCR. (F) A549 cells were transfected with pcDNA3.1-Flag-RNF149 (0.5 μg), pcDNA3.1-Flag-RNF149 (1 μg) or empty vector (0.5 or 1 μg) for 24 h and then infected with EMCV at 0.1 MOI, the cells were harvested…

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  Figure 3.. RNF149 inhibits type I IFN-mediated signaling. (A–B) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (0.5 μg), pcDNA3.1-Flag-RNF149 (1 μg), pISRE-luc (200 ng), pSTAT1-luc (200 ng), and pRL-TK (10 ng). After 24 h, the cells were treated with IFN-α or IFN-β for 12 h. Then, luminescence was detected using dual-luciferase report. (C-F) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (0.5 μg), pcDNA3.1-Flag-RNF149 (1 μg), and empty vector. After 24 h, the cells were treated with IFN-β for 12 h. Then, MX1, ISG15, OASL and ISG56 mRNA levels were detected using RT-qPCR. (G) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (1 μg) or empty vector. Then, the cells were infected with VSV-GFP at 0.01 MOI for 12 h followed by treatment with IFN-α or IFN-β for 12 h. The cells were observed microscopically. Scale bars: 300 µm. The results are presented as means ± sd. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

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  Figure 4.. RNF149 deficiency promotes JAK-STAT signaling pathway activation. (A) RNF149 knockout HEK293T cells (RNF149 KO) were constructed using CRISPR-Cas9 technology. The RNF149 protein expression was detected by Western blotting. β-actin was used as a loading control. (B–C) RNF149-KO or RNF149-WT cells infected with EMCV at 0.1 MOI were harvested at 24 hpi, RT-qPCR detect EMCV mRNA. Virus titers were analyzed by TCID50 assay. (D–E) RNF149-KO and RNF149-WT cells were transfected with pISRE-luc (200 ng), pSTAT1-luc (200 ng), or pR-TK (10 ng). After 24 h, the cells were treated with IFN-α or IFN-β for 12 h. Then, luminescence was detected using dual-luciferase report. (F) the heat map of the DEGs. (G) Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis the DGEs of upregulated in RNA-seq. The results are presented as means ± sd. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

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  Figure 5.. RNF149 inhibits the JAK-STAT signaling pathway through JAK1. (A–E) HEK293T cells were co-transfected with Myc-JAK1 (0.5 μg), Myc-TYK2 (0.5 μg), Myc-STAT1 (0.5 μg), Myc-STAT2 (0.5 μg), Myc-IRF9 (0.5 μg), pcDNA3.1-Flag-RNF149 (1 μg), pISRE-luc (200 ng), and pRL-TK (10 ng). After 24 h, luminescence was detected using dual-luciferase report. (f) HEK293T cells were co-transfected with pcDNA3.1-Flag-RNF149 (1 μg) and Myc-JAK1 (1 μg), or empty vector. After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-Myc antibodies. (G) HEK293T cells were co-transfected with EGFP-RNF149 and mcherry-JAK1. At 24 h post transfected, co-localization was performed using confocal fluorescence microscopy. The nuclei were stained by DAPI. The fluorescence intensity profile of DAPI (blue), RNF149 (green) and JAK1 (red) was measured along the line drawn by image J. Scale bars, 10 μm. The results are presented as means ± sd. ns, not significant; **, p < 0.01 versus control.

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  Figure 6.. RNF149 regulates viral replication through degrading JAK1. (A) HEK293T cells were co-transfected with pcDNA3.1-Flag-RNF149 (1 μg) or empty vector. After 24 h, the cells were treated with IFN-β for 12 h. Then, JAK1, TYK2, STAT1, STAT2, and IRF9 mRNA levels were detected using RT-qPCR. (B–F) HEK293T cells were co-transfected with Myc-JAK1 (1 μg), Myc-TYK2 (1 μg), Myc-STAT1 (1 μg), Myc-STAT2 (1 μg), Myc-IRF9 (1 μg), and pcDNA3.1-Flag-RNF149 (1 μg). After 24 h, the cell lysate was harvested. The proteins level was analyzed by Western blotting. β-actin was used as a loading control. (G) HEK293T cells were co-transfected with Myc-JAK1 (1 μg), pcDNA3.1-Flag-RNF149 (1 μg) or pcDNA3.1-Flag-RNF149 (1.5 μg). After 24 h, the cell lysate was harvested. The proteins level was analyzed by Western blotting. β-actin was used as a loading control. (H) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (1 μg) or pcDNA3.1-Flag-RNF149 (1.5 μg). After 24 h, the cell lysate was harvested. The proteins level was analyzed by Western blotting using anti-JAK1 antibodies. β-actin was used as a loading control. (i) A549 cells were transfected with negative control siRNA-ctrl, siRNA-4#. After 48 h, the…

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  Figure 7.. RNF149 RING-finger domain antagonizes IFN signaling. (A) schematic representation of the mutants of RNF149. (B) HEK293T cells were co-transfected with Flag-tag empty vector, Flag-RNF149FL (1 μg), Flag-RNF1491–221 (1 μg), Flag-RNF14967–310 (1 μg), Flag-RNF149176–400 (1 μg) and Myc-JAK1 (1 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-Myc antibodies. (C–D) HEK293T cells were co-transfected with Flag-RNF149FL (1 μg), Flag-RNF1491–221 (1 μg), Flag-RNF14967–310 (1 μg), Flag-RNF149176–400 (1 μg), pISRE-luc (200 ng), pSTAT1-luc (200 ng), and pRL-TK (10 ng). After 24 h, the cells were treated with IFN-α or IFN-β for 12 h. Then, luminescence was detected using dual-luciferase report. (e) HEK293T cells were transfected with Flag-tag empty vector, Flag-RNF149 wt (1 μg), Flag-RNF149-C269A/C272A (1 μg) or Flag-RNF149-H289A (1 μg), the proteins were analyzed by Western blotting. β-actin was used as a loading control. (f) HEK293T cells were co-transfected with Myc-JAK1 (1 μg), Flag-tag empty vector, Flag-RNF149 wt (1 μg), Flag-RNF149-C269A/C272A (1 μg) or Flag-RNF149-H289A (1 μg), the proteins were…

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  Figure 8.. RNF149 promotes the K27-linked and K33-linked ubiquitination of JAK1. (A) HEK293T cells were co-transfected with pcDNA3.1-Flag-RNF149 (1 µg) and Myc-JAK1 (1 µg). After 24 h, treated with dimethyl sulfoxide (DMSO), 3-MA (10 mM), MG-132 (10 μM), baf A1 (100 nM), and Z-VAD (20 μM) for 12 h. Protein expression was measured using Western blotting. β-actin was used as a loading control. (B) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (1 µg) or empty vector for 24 h, then cells were treated with DMSO or MG-132 for 12 h. Protein expression was measured using Western blotting. β-actin was used as a loading control. (C) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK1 (1 μg) and His-Ub (0.5 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-His antibodies. (D–E) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK1 (1 μg) and His-Ub or the indicated Ub mutant plasmids (0.5 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-His antibodies.

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  Figure 9.. The 1–559 aa region of JAK1 is critical for efficient RNF149-mediated degradation. (A) schematic representation of the mutants of JAK1. (B) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK1FL (1 μg), Myc-JAK11–559 (1 μg), Myc-JAK1560–1154 (1 μg) and Myc-JAK1851–1154 (1 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-Myc antibodies. (C) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK1FL (1 μg), Myc-JAK11–559 (1 μg), Myc-JAK1560–1154 (1 μg) and Myc-JAK1851–1154 (1 μg). After 24 h, protein expression was measured using Western blotting. β-actin was used as a loading control. (d) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK11–559 (1 μg) and the indicated Ub mutant plasmids (0.5 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-His antibodies.

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  Figure 10.. A working model of the role of RNF149 protein in the regulation of IFNs-mediated JAK-STAT signaling pathway to promote EMCV replication.