Fig 6. TRIM25 induces the ubiquitination of VP3.

(A) TRIM25 promoted the ubiquitination of VP3. The pFlag-VP3 and pHA-Ub plasmids were co-transfected with or without pMycTRIM25 into HEK293T cells for 36 h. The lysate were incubated with indicated anti-Flag mAb and detected using an IP assay. (B) Schematic representation of the constructing strategy of eight Ub mutants (K6, K11, K27, K29, K33, K48, K63, and K27R). (C and D) TRIM25 catalyzed the K27-linked polyubiquitination of VP3. pFlag-VP3 and different pHA-Ub plasmids were co-transfected with pMyc-TRIM25 into HEK293T cells for 36h. Then, lysates were incubated with indicated anti-Flag mAb and detected using IP. (E) Schematic representation of the structure of TRIM25 and TRIM25DelRING. (F) Ubiquitination of VP3 catalyzed by TRIM25 depends on its E3 Ub ligase. HEK293T cells were co-transfected with pFlag-VP3 (0.5 μg) and pMyc-TRIM25 (2 μg) or TRIM25DelRING (2 μg) plasmids for the detection of VP3 ubiquuitination by immunoblotting. (G) Degradation of VP3 induced by TRIM25 depended on its E3 Ub ligase. HEK293T cells were co-transfected with pHA-VP3 (0.5 μg) and increasing doses of TRIM25DelRING (0, 1, 2, and 4 μg) plasmids for 36 h and further detected by Western blotting. (H-J) The DF-1 cells were transfected with 2μg pMyc-TRIM25, pMyc-TRIM25DelRING, or pCAGGS at 24 h intervals and were infected with IBDV Gt strain at an MOI of 0.01. Subsequently, the cell and supernatant samples were collected at 24h p.i, respectively. (H) Expression levels of VP3 was determined using Western blotting with indicated antibodies. (I) Relative intensities of VP3 were normalized with β-actin. (J) Relative fold change of IBDV genome mRNA level of cell samples was quantified by RT-qPCR. (K) Released viral titer was detected and illustrated using a TCID₅₀ assay. All RT-qPCR results are represented as relative fold changes after being normalized to β-actin controls. Western blots were representative of one of three independently performed. Three independent experiments were performed, and data are shown as mean±standard deviations for triplicates from a representative experiment.*, P < 0.05; **, P < 0.01; ns, no significant difference.