Virologica Sinica|中山大学郭德银/吴俊玉/李春梅/曹流揭示TRIM25在哺乳动物胚胎干细胞中的抗病毒作用机制|ifn|中山大学郭德银|免疫|吴俊玉|哺乳动物|基因改造病毒|抗病毒|曹流|李春梅

https://doi.org/10.1016/j.virs.2026.07.011

摘要

哺乳动物胚胎干细胞（ESCs）尽管缺乏功能性干扰素（IFN）信号传导，却对病毒感染具有高度抗性。然而，其抗病毒防御机制的基础尚未完全阐明。本研究探讨了TRIM25——一种已知的IFN依赖性先天性免疫关键驱动因子——在IFN缺陷型小鼠ESCs（mESCs）中的抗病毒功能作用及分子机制。TRIM25在mESCs中展现出广谱抗病毒活性。通过RNAi敲低或CRISPR介导的Trim25基因敲除实验发现，该因子缺失显著促进了EMCV和VSV病毒的复制与传播。从机制层面看，TRIM25在mESCs中的抗病毒活性与IFN产生及基础ISG表达均无关。在病毒感染的mESC中，TRIM25从细胞核转移到细胞质，直接结合病毒RNA，与G3BP1形成细胞质凝聚物，在感染过程中与dsRNA病灶共定位。G3BP1的遗传扰动同样损害了mESC的抗病毒防御，揭示了TRIM25和应激颗粒成分在限制病毒复制方面的重要协同作用。我们的研究将TRIM25确定为ESC中的关键RNA传感效应器，描绘了一个以前未被认识的IFN-非依赖性内在免疫轴，该轴将病毒RNA监测与应激颗粒介导的抑制联系起来，从而扩展了多能细胞自主抗病毒策略的范式。

Fig. 1. Depletion of Trim25 promotes EMCV proliferation in 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.

Fig. 2. Knockout of Trim25 significantly enhances the replication of EMCV in ESCs. A, B Trim25-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 of EMCV infection (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.

Fig. 3.Knockout of Trim25 Enhances VSV Proliferation in 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.

Fig. 4. TRIM25 does not affect IFN pathway and the expression of intrinsic ISGs in mESCs. A, B Trim25-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.

Fig.5. TRIM25 binds viral RNAs in 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.

Fig. 6. TRIM25 interacts with G3BP1 in viral-infected 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.

Fig. 7. G3BP1 collaborates with TRIM25 to inhibit viral infection in 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.

Fig 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.

Fig. 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.

Fig. 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.

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