Fig. 2.

The T₅₅ residue of RIG-I is critical for TRIM25 binding. (A) T₅₅I mutation abolishes RIG-I-TRIM25-interaction. At 48 h after transfection with GST, GST-RIG-I 2CARD WT, or GST-RIG-I 2CARD T₅₅I together with TRIM25-V5 WCLs were used for GST-PD, followed by IB with α-V5 or α-GST. Arrows indicate the ubiquitinated bands. (B and C) T₅₅I mutation abolishes RIG-I CARD ubiquitination and downstream signaling. GST-RIG-I 2CARD WT or GST-RIG-I 2CARD T₅₅I with or without TRIM25-V5 was expressed in HEK293T. WCLs were used for GST-PD, followed by IB with α-GST or α-Ub. Arrows indicate the ubiquitinated bands. (C) GST-RIG-I 2CARD WT or T₅₅I with or without TRIM25 together with IFN-β luciferase and pGK-β-gal were expressed in HEK293T cells as described in Fig. 1D. Data represent the mean ± SD (n = 3). (D) T₅₅I mutation strongly decreases RIG-I binding to MAVS. HEK293T were transfected with MAVS-CARD-PRD-Flag together with GST or GST-RIG-I 2CARD fusion constructs. WCLs were subjected to GST-PD, followed by IB with α-Flag, α-Ub, or α-GST. MAVS-CARD-PRD expression was determined by IB with α-Flag. (E and F) Ubiquitination, TRIM25 binding, and signaling activity of RIG-I 2CARD T₅₅ mutants. GST or GST-RIG-I 2CARD was expressed in HEK293T cells. WCLs were subjected to GST-PD, followed by IB with α-Ub, α-GST, or α-TRIM25. Arrows indicate the ubiquitinated bands. Luciferase assay was performed as described in Fig. 1D. Data represent the mean ± SD (n = 3).