Fig 1: The MGF-505-7R-deficient ASFV increased the JAK-STAT1 signaling pathway.A, diagram indicating the position of the MGF-505-7R open reading frame in the ASFV CN/GS/2018 genome. The donor plasmid with the homologous arms to the ASFV CN/GS/2018 and eGFP under control of the p72 promoter in the orientation as indicated. The final genomic changes introduced to develop the ASFV-Δ7R where the sequence of the donor plasmid eGFP reporter was introduced to replace the ORF of the MGF-505-7R as indicated. B, the successful recombination was confirmed using a fluorescence microscope. C, the absence of parental CN/GS/2018 was confirmed using PCR. D, effects of the ASFV MGF-505-7R knockout (ASFV-Δ7R) on the expression of the endogenous ASFV MGF-505-7R. E, replication of the ASFV-Δ7R in Ma-104 cells. Ma-104 cells were infected with the parental ASFV or ASFV-Δ7R (MOI: 0.01) for the indicated time before RT-PCR analysis. Data represent the means from three independent experiments. F, effects of the ASFV or ASFV-Δ7R on IFN-γ-induced Irf1 transcription. PAMs were infected with the parental ASFV or ASFV-Δ7R (MOI: 0.01) for 21 h. The cells were untreated or treated with IFN-γ (10 ng/ml) for 3 h before RT-PCR analysis. Data represent the means from three independent experiments. G, effects of the ASFV-Δ7R on the IFN-γ-induced phosphorylation of downstream components. PAMs were infected with the parental ASFV or ASFV-Δ7R for the indicated times and were then treated or untreated with IFN-γ for 3 h. H, effects of ASFV-Δ7R on JAK1 and JAK2, Hes5, P30, and RNF125 expression. The cells were infected with the parental ASFV or ASFV-Δ7R for the indicated times before western blot analysis. I, effects of the ASFV or ASFV-Δ7R on the interaction between RNF125 and JAK1. PAMs were infected with the ASFV or ASFV-Δ7R (MOI: 0.01) for 12 h before coimmunoprecipitation and western blot analyses. J, effects of the ASFV or ASFV-Δ7R on the interaction between Hes5 and JAK2. The experiments were similarly performed as in I.
Fig 2: The ASFV MGF-505-7R degrades JAK1 by upregulating RNF125 expression and JAK2 by downregulating Hes5 protein.A, MGF-505-7R enhanced JAK1 ubiquitination. First, 293T cells (2 × 105) were transfected with Myc-JAK1 (1 μg), HA-Ub (0.5 μg), and Flag-MGF-505-7R (1 μg) for 24 h. Coimmunoprecipitation and western blot analyses were performed with the indicated antibodies. B, coimmunoprecipitation and western blot analyses for the detection of the endogenous polyubiquitination of JAK1 in the ASFV- or ASFV-Δ7R-infected PAMs for the indicated times. PAMs were infected with the ASFV or ASFV-Δ7R (MOI: 0.01) for the indicated times. Coimmunoprecipitation and western blot analyses were performed with the indicated antibodies. C, MGF-505-7R interacted with RNF125 in the overexpression system. The 293T cells (2 × 106) were transfected with the indicated plasmids (5 μg each). Coimmunoprecipitation and western blot analyses were performed with the indicated antibodies. The levels of the transfected proteins were analyzed using western blotting with anti-HA or -Flag antibodies. D, endogenous associations between MGF-505-7R and RNF125. PAMs were infected with the ASFV for the indicated times before coimmunoprecipitation and western blot analyses. E, MGF-505-7R increased the expression of the RNF125 protein. First, 293T cells (2 × 105) were transfected with HA-RNF125 (1 μg) and Flag-MGF-505-7R (0, 0.25, 0.5, and 1.0 μg) for 24 h. Cell lysates were analyzed using western blotting with the indicated antibodies. F, RNF125 inhibited the expression of JAK1 protein. The experiments were similarly performed as in E. G, RNF125 increased the MGF-505-7R degradation of JAK1. The 293T cells (2 × 105) were transfected with the indicated plasmids for 24 h. Cell lysates were analyzed using western blotting with the indicated antibodies. H, MGF-505-7R interacted with Hes5. The experiments were similarly performed as in A. I, endogenous associations between MGF-505-7R and RNF125. The experiments were similarly performed as in D. J, Hes5 increased the expression of JAK2 protein. The experiments were similarly performed as in E. K, MGF-505-7R inhibited the expression of Hes5 protein. The experiments were similarly performed as in E. L, MGF-505-7R inhibited the Hes5-mediated expression of JAK2. The experiments were similarly performed as in G. M and N, the effect of RNF125 or Hes5 knockdown on JAK1 or JAK2 expression. The PAMs were transfected with Con-RNAi, RNF125-RNAi, or Hes5-RNAi (1.0 μg/ml) for 48 h. Then, the cells were infected with the ASFV (MOI: 0.01) for the indicated time. Cell lysates were analyzed using western blotting with the indicated antibodies. Con-RNAi, control-RNAi.
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