Fig 1: Knocking down TRIM25-R54P-specific interactors identifies essential substrates for TRIM25 antiviral activity.(A-B) TRIM25 inducible cells were transfected with pooled siRNAs for either (A) hits specific to TRIM25-R54P in the absence of viral infection or (B) hits specific to TRIM25-R54P in the presence of viral infection. Cells were induced for TRIM25-WT expression at 1 μg/mL dox, infected with Toto1101/Luc at an MOI of 0.01 PFU/cell, and lysed at 24 h.p.i. for measurement of luciferase activity. Asterisks indicate statistically significant differences as compared to the NT pool siRNA (One-way ANOVA, Dunnett’s multiple comparison test; **, p<0.01; ****, p<0.0001). Unlabeled comparisons are not significant. Data are either (A) pooled from or (B) representative of two independent experiments. (C-F) Parental 293T cells (TRIM25: endogenous) or TRIM25 inducible (TRIM25: inducible) cells were transfected with individual siRNAs for (C,E) NME1 or (D,F) PABPC4, induced for TRIM25-WT expression at 1 μg/mL dox, and (C-D) had RNA extracted for RT-qPCR analysis or (E-F) infected with Toto1101/Luc at an MOI of 0.01 PFU/cell. Cells were lysed at 24 h.p.i. for measurement of luciferase activity. Asterisks indicate statistically significant differences as compared to the NT pool for each cell line. 293T and TRIM25-WT inducible cell lines were statistically analyzed independently from one another (One-way ANOVA, Dunnett’s multiple comparison test; *, p<0.05; ****, p<0.0001). Data are representative of two independent experiments for each cell line.
Fig 2: TRIM25 interacts with and polyubiquitinates NME1.(A) Western blot of TRIM25 KO and TRIM25 inducible cells transfected with myc-tagged UPF1 or NME1 in the presence of 1 μg/mL dox. Lysates were subjected to a FLAG IP. Data are representative of two independent experiments. (B) Western blot of TRIM25 inducible cells transfected with myc-NME1 in the presence or absence of 1 μg/mL dox. Lysates were subjected to a myc IP. Data are representative of two independent experiments. (C) Western blot of TRIM25 KO and TRIM25 inducible cells in the presence of 1 μg/mL dox. Lysates were subjected to a FLAG IP. Data are representative of two independent experiments. (D) Western blot of TRIM25 KO and TRIM25 inducible cells treated with 1 μg/mL dox and transfected with myc-NME1 and HA-Ub-WT. Lysates were subjected to myc IP. Data are representative of three independent experiments.
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