Figure 6.

Laboratory of genetics and physiology 2 (LGP2) inhibits downstream signaling of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). (A,B) LGP2 inhibits RIG-I-mediated IFN-β promoter stimulator 1 (IPS-1) and mediator of IRF3 activation (MITA) promoter activities. Fathead minnow (FHM) cells were transiently transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of RIG-I expression plasmid, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc for 16 h and then infected with grass carp reovirus (GCRV). Luciferase activities were conducted at 24 h after GCRV infection. (C,D) LGP2 inhibits MDA5-mediated IPS-1, but not MITA promoter activity. FHM cells were transiently transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of MDA5 expression plasmids, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc in 24-well plates. At 16 h post-transfection, the cells were infected with GCRV for 24 h and then subjected to luciferase activities analysis. (E) Upper: schematic representations of full-length RIG-I and the three domains constructed in the present study. Below: LGP2 interacts with RIG-I-Helicase, RIG-I-RD, but not RIG-I-CARDs domain. FHM cells were cotransfected with 4 µg LGP2-Flag and 4 µg RIG-I-CARD-HA or RIG-I-Helicase-HA or RIG-I-RD-HA for 24 h in 10 cm² dishes. Co-IP was performed using anti-HA antibody (Ab), and mouse IgG was used as control. IPs were analyzed by IBs with anti-HA and anti-Flag, respectively. Expression of LGP2-Flag (input) was examined with anti-Flag. (F) Upper: full-length MDA5 and its domain structures. Below: LGP2 interacts with MDA5-CARDs, MDA5-Helicase, and MDA5-RD. FHM cells were transfected with the indicated plasmids (4 µg each). Twenty-four hours later, cells were lysed, Co-IP and IB analyses were performed with the indicated Abs. (G) LGP2 inhibits RIG-I-CARDs-mediated MITA, but not IPS-1 promoter activity. FHM cells were transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of RIG-I-CARD-HA expression plasmid, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc. Luciferase assays were performed at 24 h post-transfection. (H) LGP2 inhibits MDA5-CARDs-mediated IPS-1 and MITA promoter activities. FHM cells were transfected with 200 ng of LGP2-Flag or empty vector, 200 ng of MDA5-CARD-HA expression plasmid, 30 ng of pRL-TK plus 200 ng of IPS-1pro-luc or MITApro-luc. Luciferase assays were performed at 24 h post-transfection. Error bars indicate SD (n = 4). Asterisks indicate significant differences from control (**0.001 < P < 0.01; ***P < 0.001).