The antiviral role of TRIM25 in mammalian embryonic stem cells

Figures

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  Fig. 1. Depletion of Trim25 promotes EMCV proliferation in embryonic stem cells (ESCs). A, B The Trim25 was depleted in D3 (A) or E14TG2a (B) cells by siRNAs and the RNA levels of Trim25 and EMCV were detected by RT-qPCR. C, D Cells were infected with EMCV after transfection with siRNAs for 24 h. The protein level of EMCV-VP1 were measured by Western blotting in Trim25-knockdown D3 (C) or E14TG2a (D) cells at 48 hpi. Actin was used as the loading control. E, F The infected D3 (E) or E14TG2a (F) cells were visualized by immunofluorescence staining with anti-VP1 antibody at 48 hpi of EMCV infection (MOI = 1). VP1 (red) and DAPI (blue). Scale bar, 20 μm. G, H The viral titers in the medium of Trim25-knockdown D3 (G) or E14TG2a (H) cells at 48 hpi were measured by plaque assay. Data are mean ± SEM. Two-tailed unpaired Student’s t-test was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Fig. 2. Knockout of Trim25 significantly enhances the replication of EMCV in embryonic stem cells (ESCs). A, BTrim25-knockout D3 (A) or E14TG2a (B) cells were infected EMCV (MOI = 1). The protein levels of EMCV-VP1 and TRIM25 were detected by Western blotting at 48 hpi. C, D The viral RNA levels of EMCV in the Trim25-knockout D3 (C) or E14TG2a (D) cells were measured by RT-qPCR at 48 hpi of EMCV infection. E–H The EMCV-infected D3 (E) or E14TG2a (G) cells were analyzed by flow cytometry after staining with anti-VP1 antibody at 48 hpi (MOI = 1). Quantitative analysis of the flow cytometry results were shown in (F and H). I, J The viral titers in the medium of Trim25-knockout D3 (I) or E14TG2a (J) cells at 48 hpi were measured by plaque assay. K, L The plasmids expressing GFP or TRIM25-GFP were transfected into E14TG2a- Trim25-KO-2 cell line followed by EMCV infection (MOI = 1). At 24 hpi, the levels of viral RNA were measured by RT-qPCR (K) and the EMCV-VP1 protein levels were analyzed by Western blotting (L). Data are mean ± SEM. Two-tailed unpaired Student’s t-test was used for statistical analysis. ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Fig. 3. Knockout of Trim25 enhances VSV proliferation in embryonic stem cells (ESCs). A The control and Trim25-knockout E14TG2a cells were infected with VSV-GFP virus at 0.01 MOI. The viral RNA levels of VSV were measured by RT-qPCR at the indicated time points after virus infection. B The protein levels of VP1 were detected by Western blotting at the indicated time points after virus infection. L.E., long exposure. S.E., short exposure. C–E The control and Trim25-knockout E14TG2a cells were infected with VSV-GFP (MOI = 0.01) for 18 h and the GFP-positive cells were visualized by fluorescence microscopy (C) or were quantitatively analyzed by flow cytometry (D and E). F The VSV viral titers in the medium of Trim25-knockout E14TG2a cells at the indicated time points were measured by plaque assay. Data are mean ± SEM. Two-tailed unpaired Student’s t-test was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Fig. 4. TRIM25 does not affect IFN pathway and the expression of intrinsic ISGs in mouse embryonic stem cells (mESCs). A, BTrim25-knockdown D3 (A) or E14TG2a (B) cells were infected with EMCV (MOI = 1). The mRNA levels of Ifnb, Isg15 and Mx1 were measured by RT-qPCR at 24 hpi. C The mRNA levels of the intrinsically expressed ISGs were measured by RT-qPCR in Trim25-depleted D3 cells at 24 hpi of EMCV infection. D, E The E14TG2a (D) or D3 (E) cells were infected with EMCV. The mRNA levels of the constitutive ISGs were measured by RT-qPCR in the mock or EMCV infected cells at 24 hpi. Data are mean ± SEM. Two-tailed unpaired Student’s t-test was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, and n.s. = not significant.

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  Fig. 5. TRIM25 binds viral RNAs in embryonic stem cells (ESCs). A Colocalization of TRIM25 and dsRNA in E14TG2a cells infected with EMCV at 48 hpi. TRIM25 (green), dsRNA (red) and DAPI (blue). Scale bar, 5 μm. B, C TRIM25 binds with viral RNA. EMCV-infected D3 (B) or E14TG2a (C) cells were lysed and immunoprecipitated with anti-TRIM25 antibody and the enrichment of viral RNA were detected by RT-qPCR assay. D, E VSV-infected D3 (D) or E14TG2a (E) cells were lysed and immunoprecipitated with anti-TRIM25 antibody and the enrichment of viral RNA were detected by RT-qPCR assay. Data are mean ± SEM. Two-tailed unpaired Student’s t-test was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Fig. 6. TRIM25 interacts with G3BP1 in virus-infected embryonic stem cells (ESCs). A Colocalization of TRIM25 and G3BP1 in E14TG2a cells infected with EMCV at 48 hpi. TRIM25 (green), G3BP1 (red) and DAPI (blue). Scale bar, 5 μm. B G3BP1 binds to TRIM25. EMCV infected E14TG2a cell lysates were immunoprecipitated by anti-G3BP1 antibody. The enrichment of G3BP1 and TRIM25 were analyzed by Western blotting. Normal IgG was used as negative control. C Colocalization of viral dsRNA and G3BP1 in E14TG2a cells infected with EMCV at 48 hpi. dsRNA (green), G3BP1 (red) and DAPI (blue). Scale bar, 5 μm. Manders' colocalization coefficient of dsRNA with G3BP1 were measured by using Image J software with Coloc 2 plugin. D, E G3BP1 binds with viral RNA. EMCV-infected D3 (D) or E14TG2a (E) cells were lysed and immunoprecipitated with anti- G3BP1 antibody and the enrichment of viral RNA were detected by RT-qPCR assay. Data are mean ± SEM. Two-tailed unpaired Student’s t-test was used for statistical analysis. ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Fig. 7. G3BP1 collaborates with TRIM25 to inhibit viral infection in embryonic stem cells (ESCs). A, B The G3bp1 was depleted in D3 (A) or E14TG2a (B) cells by siRNAs and the mRNA levels of Oct4 and G3bp1 were detected by RT-qPCR. C–H Cells were infected with EMCV after transfection with siRNAs for 24 h. The viral RNA levels were measured by RT-qPCR in G3bp1-knockdown D3 (C) or E14TG2a (D) cells at 24 hpi. And the protein level of VP1 were measured by Western blotting in G3BP1-knockdown D3 (E) or E14TG2a (F) cells at 48 hpi. The viral titers in the medium of G3bp1-knockdown D3 (G) or E14TG2a (H) cells at 48 hpi were measured by plaque assay. I–K The siRNAs (siNC or siG3bp1) were transfected into the E14TG2a-WT or E14TG2a-Trim25-KO-2 cell lines followed by EMCV infection. At 24 hpi, the RNA levels of G3bp1 (I) and EMCV (J) were measured by RT-qPCR. The EMCV-VP1 protein levels were analyzed by Western blotting (K). Data are mean ± SEM. Two-tailed unpaired Student’s t-test was used for statistical analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and n.s. = not significant.

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  Supplementary Figure S1. Knockout of Trim25 does not affect the pluripotency of ESCs. A, B The expression levels of the pluripotent markers (Oct4 and Nanog) in the Trim25-knockout D3 (A) or E14TG2a (B) cells were measured by RT-qPCR. Data are mean ± SEM. Two-tailed unpaired Student’s t test was used for statistical analysis. *P < 0.05, and ns = not significant. C-F The wild-type and Trim25-knockout D3 (C and D) or E14TG2a (E and F) cells were fixed and stained with pluripotent markers: Oct4 (C and E) and SSEA-1 (D and F). Nuclei were stained with DAPI (blue). Scale bar, 10 μm.

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  Supplementary Figure S2. Virus infection is deficient to induce IFNβ expression in ESCs. D3, E14TG2a and MEF cells were infected with EMCV at 1 moi. The mRNA levels of Ifnb were measured by RT-qPCR at 24 hpi. Data are mean ± SEM. Two-tailed unpaired Student’s t test was used for statistical analysis. ∗∗∗P < 0.001, and ns = not significant.

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  Supplementary Figure S3. Depletion of G3bp1 shows minimal effects on the pluripotency of ESCs. The D3 (A and B) or E14TG2a (C and D) cells transfected with siRNAs (siNC or siG3bp1) were fixed and stained with pluripotent markers: Oct4 (A and C) and SSEA-1 (B and D). Nuclei were stained with DAPI (blue). Scale bar, 10 μm.