Fig 2: miR-30a inhibits the expression of TRIM25 by targeting the 3'UTR of TRIM25. (A) Computational prediction of potential miR-30a target sites in the 3'UTR of TRIM25. The predicted target sites of miR-30a are underlined and were mutated as indicated. The fragments were inserted in XbaI site. (B) Luciferase activity in lysates of 293T cells co-transfected with TRIM25-3'UTR or TRIM25-3'UTR-mutant luciferase reporter vector and miR-30a mimics or NC mimics. (C–E) 293T cells were transfected with miR-30a or NC mimics (80 nM) for 24 h. Cells were analyzed by RISC-IP followed by qRT-PCR to analyze TRIM25 (normalized to GADPH) (C), or miR-30a compared to RNAs extracted from total samples of lysates from 293T cells (normalized to U6) (D) or WB to analyze Ago2 expression from input (E). (F) qRT-PCR analysis of TRIM25 mRNA levels in 293T cells transfected with miR-30a mimics or miR-30a inhibitor (80 nM). Data are presented as mean ± SD. Statistical significance was analyzed using t-tests. **P < 0.01; ***P < 0.001. (G) WB analysis of TRIM25 expression levels in cells transfected with miR-30a mimics at the indicated doses (20–80 nM). ß-actin expression was analyzed as a loading control.

Fig 3: TRIM25 restricts CVB3 replication by upregulating IFN-ß activation and production. (A) HeLa cells were transiently transfected with pCAGGS-TRIM25-Myc or pCAGGS. 24 hpt, cells were subjected to WB for detecting TRIM25 expression levels. (B, C) HeLa cells were transiently transfected with pCAGGS-TRIM25-Myc or pCAGGS. 24 hpt, cells were infected with CVB3 at an MOI of 0.1. Supernatants were collected and subjected to TCID50 (B) and WB analyzed VP1 levels (C). (D) HeLa cells were transfected with control siRNA (si-con) or TRIM25 siRNA (si-TRIM25) and the knockdown efficiency was analyzed by WB at 24 hpt. (E, F) Cells were transfected with si-con or si-TRIM25 (50 nM) for 24 h and infected with CVB3 (MOI = 0.1). Cells and supernatants were harvested at the indicated times. (E) TCID50 assay and (F) WB analysis evaluated CVB3 replication. (G) IFN-ß promoter vector along with pRL-TK were co-transfected with pCAGGS-TRIM25-Myc or pCAGGS into cells for 24 h, followed by stimulation with poly(I:C) (10 µg/ml) for 12 h. Then, cells were harvested for luciferase assay. (H) qRT-PCR analysis of IFN-a, IFN-ß, MX1, and ISG15 expression in cells transfected with pCAGGS-TRIM25-Myc or pCAGGS for 24 h and then infected with CVB3 for 24 h at an MOI of 0.1. Data were normalized to GAPDH expression and are the mean ± SD of three independent experiments. Statistical significance was analyzed using t tests. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: miR-30a suppresses TRIM25-mediated RIG-I activation and IFN production. (A) pCAGGS-RIG-I-HA and pCAGGS-TRIM25-Myc were co-transfected with or without miR-30a. The interaction was probed by immunoprecipitation. (B) 293T cells were co-transfected with the indicated plasmids and miR-30a or NC mimics for 36 h, and were treated with or without poly(I:C) (10 µg/ml) for 12 h. Anti-Flag immunoprecipitates prepared from the cell extracts were analyzed by WB using the indicated antibodies. (C, D) 293T cells seeded in 24-well plates were co-transfected using the IFN-ß-Luc firefly luciferase reporter plasmid and the Renilla luciferase control reporter plasmid pRL-TK. For the experiment, pCAGGS-RIG-I-Flag or pCAGGS-2CARD-Flag were co-transfected with or without miR-30a. (E) pCAGGS-2CARD-Flag (0.25 µg) and pCAGGS-TRIM25-Myc (0.5 µg) plasmids with or without miR-30a were co-transfected. The luciferase activity in cell lysates was measured. The data are presented as the mean ± SD from three experiments. Statistical significance was analyzed using t tests. *P < 0.05; **P < 0.01. (F) Ubiquitin-HA (0.5 µg), pCAGGS-2CARD-Flag (0.5 µg), or pCAGGS-TRIM25-Myc (1.0 µg) were co-transfected with or without miR-30a. 24 hpt, cells were treated with poly(I:C) (10 µg/ml) for 12 h, cell lysates were analyzed by immunoprecipitation using the indicated antibodies to detect the ubiquitination of RIG-I-2CARD.

Fig 5: Interaction between the HRSV NS1 protein and the RIG–I CARD/TRIM25 complex. (A) HEK293T cells were transfected with the indicated plasmids. Cells were lysed in lysis buffer 24 h post–transfection and subjected to immunoblotting (IB) analysis with anti–RIG–I, anti–V5, or anti–HA antibodies. (B, C) HEK293T cells were transfected with the indicated plasmids. Cells were lysed in lysis buffer 24 h post–transfection and subjected to immunoprecipitation (IP) with an anti–V5 antibody, followed by IB with an anti–FLAG antibody. A portion of the cell lysates was also subjected to IB. HRSV, human respiratory syncytial virus.