Fig 1.
Individual TRIM25 RING residues required for TRIM25 autoubiquitination.
(A) Alignment of the RING E3 ligases MDM2 (dark blue) and TRIM25 (light blue) in complex with ubiquitin (red) and the E2 UbcH5 (gray), performed using UCSF Chimera [30]. Highlighted in gold (TRIM25) and yellow (MDM2) are homologous residues. PDB: 5MNJ (MDM2), 5EYA (TRIM25). (B) Western blot of 293T cells transfected with FLAG-TRIM25 mutants and HA-ubiquitin (Ub). Lysates were subjected to FLAG IP. Data representative of three independent experiments.
Fig 2.
TRIM25 co-IP/MS identifies TRIM25 interactors.
(A) Western blot of TRIM25-WT and -R54P doxycycline (dox)-inducible 293T cell lines in the presence of increasing amount of dox (0, 0.001, 0.01, 0.1, 1, and 10 μg/mL). Data are representative of two independent experiments. (B) Schematic of co-IP/MS experiment to identify TRIM25 interactors. (C-D) Volcano plots of proteins significantly enriched over TRIM25 KO background in TRIM25-R54P co-IP/MS in the (C) absence or (D) presence of viral infection. Data representative of two independent experiments. Blue dots represent proteins that were also significantly enriched in TRIM25-WT co-IP and red dots represent proteins that were only enriched in TRIM25-R54P co-IP. Proteins were counted as enriched when log2FC>1.5 and -log10Pvalue>1.3 (Pvalue<0.05). The R package EnhancedVolcano [32] was used to generate volcano plots. (E) Gene ontology terms significantly enriched in all unique TRIM25-WT and TRIM25-R54P interactors. Analysis performed for GO terms in biological processes using DAVID [33,34].
Table 1.
TRIM25-R54P interactors in the absence of virus.
Interactors pulled down in both independent experiments shown here; proteins also enriched in TRIM25-WT co-IP are italicized and bolded. Fold change = FC. In EXP #2, the “i” prefacing log2FC and Pvalue refers to how missing data values were imputed.
Table 2.
TRIM25-WT interactors in the absence of virus.
Interactors pulled down in both independent experiments shown here; proteins also enriched in TRIM25-R54P co-IP in both independent experiments are italicized and bolded. Fold change = FC. In EXP #2, the “i” prefacing log2FC and Pvalue refers to how missing data values were imputed.
Table 3.
TRIM25-R54P interactors during viral infection.
Interactors pulled down in both independent experiments shown here; proteins also enriched in TRIM25-WT co-IP in both independent experiments are italicized and bolded. Fold change = FC. In EXP #2, the “i” prefacing log2FC and Pvalue refers to how missing data values were imputed.
Table 4.
TRIM25-WT interactors during viral infection.
Interactors pulled down in both independent experiments shown here; proteins also enriched in TRIM25-R54P co-IP in both independent experiments are italicized and bolded. Fold change = FC. In EXP #2, the “i” prefacing log2FC and Pvalue refers to how missing data values were imputed.
Fig 3.
TRIM25 interacts with and polyubiquitinates G3BP.
(A) Western blot of TRIM25 KO 293T cells transfected with myc-G3BP1/2 and FLAG-TRIM25-WT, -R54P, or -PTAA. Lysates were subjected to FLAG IP. Data are representative of three independent experiments. (B) Western blot of TRIM25 KO and TRIM25-WT, -R54P, or -PTAA inducible cells transfected with myc-G3BP1/2 and HA-Ub-WT in the presence of 1 μg/mL dox. (C) Western blot of TRIM25-WT inducible cells transfected with myc-G3BP1/2 and HA-Ub-WT, -K48, or -K63 in the presence of 1 μg/mL dox. (B-C) Lysates were subjected to myc IP. Data are representative of three independent experiments.
Fig 4.
TRIM25 interacts with and mono-ubiquitinates UPF1.
(A) Western blot of TRIM25 inducible cells transfected with V5-tagged UPF1 in the presence or absence of 1 μg/mL dox. Lysates were subjected to FLAG IP. Data are representative of three independent experiments. (B-C) Western blot of TRIM25 inducible cells transfected with (B) V5-UPF1 or (C) V5-UPF1 WT and mutants (K281R, K592R) and HA-Ub in the presence of 1 μg/mL dox. Lysates were subjected to V5 IP. Data are representative of two independent experiments for (B) and of three independent experiments for (C).
Fig 5.
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.
Fig 6.
TRIM25 interacts with and polyubiquitinates PABPC4.
(A) Western blot of TRIM25 KO and TRIM25 inducible cells transfected with myc-tagged PABPC4 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 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. (C) Western blot of TRIM25 KO and TRIM25 inducible cells treated with 1 μg/mL dox and transfected with myc-PABPC4 and HA-Ub-WT. Lysates were subjected to myc IP. Data are representative of two independent experiments. (D) Western blot of TRIM25-WT inducible cells treated with 1 μg/mL dox and transfected with myc-PABPC4 and HA-Ub-WT, -K48, or -K63. Lysates were subjected to myc IP. Data are representative of two independent experiments.
Fig 7.
Point mutation in TRIM25 RING domain cripples TRIM25 antiviral activity.
(A-C) Dox inducible TRIM25-WT or -R54P cells were induced for TRIM25-WT or -R54P expression at 1 μg/mL dox. Cells were infected with (A) SINV Toto1101/Luc at an MOI of 0.01 plaque forming unit (PFU)/cell, and lysed at 6, 12, 24, 32, and 40 hours post infection (h.p.i.); data combined from three independent experiments, error bars indicate range; or (B) Sindbis virus (SINV) Toto1101 at an MOI of 0.01 PFU/cell, harvesting supernatant at 6, 12, 24, 32, and 40 h.p.i. for plaque assays; data representative of two independent experiments, error bars indicate range; or (C) SINV Toto1101/Luc:ts6 at an MOI of 1 PFU/cell and lysed at 6 h.p.i. for measurement of luciferase activity; data representative of two independent experiments, error bars indicate standard deviation. (D) Percent infected cells (GFP+) at MOI of 0.01 PFU/cell (SINV 24 h.p.i.; Ross River virus (RRV) 24 h.p.i.; o’nyong-nyong virus (ONNV) 22 h.p.i.; Venezuelan equine encephalitis virus (VEEV) 10 h.p.i.) were normalized to that of the respective cell line without dox (set to one-fold). Asterisks indicate statistically significant differences, calculated using (A-B, D) Two-way ANOVA and Tukey’s multiple comparisons test: **, p<0.01; ***, p<0.001; ****, p<0.0001; (light blue compares WT +/- dox, dark blue compares R54P +/- dox) or (C) Two-way ANOVA and Šidák’s multiple comparisons test: ****, p<0.0001. Data for each virus (demarcated by dashed lines) was statistically analyzed independently. (E) TRIM25 inducible cells were treated with poly(I:C) in the presence or absence of dox, and RNA was harvested for RT-qPCR analysis. mRNA levels of IFN/ISGs in TRIM25-WT or R54P were normalized to that of the respective cell line without dox (set to one-fold, horizontal dotted line). Data representative of two independent experiments. mRNA fold change for each gene (demarcated by vertical dashed lines) was statistically analyzed independently. Asterisks indicate statistically significant differences as compared to the -dox condition (Two-way ANOVA and Šidák’s multiple comparisons test: *, p<0.05; ***, p<0.001; ****, p<0.0001).
Fig 8.
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.
Table 5.
Key resources.