Fig. 4

LGP2 functions as a dual regulation of itself- and virus-induced IFN response

(A and B) LGP2 played antithetic role in itself-induced fish IFN promoter activation (A) and upregulation of IFN-inducible proteins (B). EPC cells seeded in 24-well plates (A) or 6-well plates (B) were transfected with LGP2 at the indicated doses for 48 h, followed by luciferase assays (A) or by western blot detection of PKR, IRF3, and LGP2 with corresponding antibodies (B). Western blot in (A) showed the ectopically expressed LGP2 in a dose-dependent manner by zebrafish LGP2 antibody.

(C and D) IRF3 dimerization was analyzed in SVCV-infected CAB cells in the absence or presence of LGP2. CAB cells seeded in 6-well plates overnight were transfected with LGP2 at the indicated doses for 24 h, followed by infection with SVCV at a relatively low titer (2×10⁵ TCID₅₀/mL) (C) or at a relatively high titer (2×10⁶ TCID₅₀/mL) (D) for another 24 h. The transfected cells were collected to detect IRF3 dimerization by native PAGE with anti-IRF3 Ab.

(E and F) LGP2 exerted an early synergizing and a late inhibitory regulation on ifn, viperin, and irf3 mRNA expression along SVCV infection. EPC cells seeded in 6-well plates overnight were transfected with the indicated doses of LGP2 for 24 h, followed by infection with SVCV (2×10⁵ TCID₅₀/mL). At the indicated time postinfection, cells were collected to detect IRF3 dimerization by native PAGE with anti-IRF3 Ab and LGP2 expression by SDS-PAGE with anti-LGP2 Ab (E) or to detect the expression of lgp2, ifn, viperin, and irf3 expression by RT-qPCR (F). Data were shown as mean ± SD (N = 3). P values were calculated using Student’s t test. ∗p < 0.05, ∗∗p < 0.01.