Fig. 2

Overexpression of TMEM47 blocks IFN induction. (A) TMEM47 inhibits the expression of epc ifn, epc vig1, epc isg15-1, epc irf7, and epc rig-i induced by SVCV. EPC cells seeded in six-well plates overnight were transfected with 2 µg TMEM47-Myc or empty vector and treated with SVCV (MOI = 1) at 24 h post-transfection. At 24 h after stimulation, total RNAs were extracted for further qPCR assays. (B) TMEM47 blocks the expression of cgIFN-A, cgIFN-B, cgVig-A, cgVig-B, cgRIG-A, and cgRIG-B induced by CyHV-2. Gibel carp brain (GiCB) cells seeded in six-well plates overnight were transfected with 2 µg TMEM47-Myc or empty vector and infected with CyHV-2 (MOI = 0.001) at 24 h post-transfection. At 48 h after stimulation, total RNAs were extracted for further qPCR assays. (C and D) TMEM47 suppresses poly I:C/poly dA:dT-induced IFNφ1pro/ISRE activation in a dose-dependent manner. EPC cells were seeded in 24-well plates overnight and then transfected with 250 ng IFNφ1pro-Luc (C) or ISRE-Luc (D) and 50 ng pRL-TK, plus 250 ng pcDNA3.1-TMEM47 (250 ng or 100/250/500 ng) or pcDNA3.1(+) (control vector). At 24 h post-transfection, cells were untreated (null) or transfected with poly I:C (0.5 µg) or poly dA:dT (0.5 µg). Luciferase activities were monitored at 24 h after stimulation. The promoter activity is presented as relative light units (RLU) normalized to Renilla luciferase activity. (E) Effects of TMEM47 RNA interference (RNAi) on the expression of endogenous TMEM47. EPC cells were seeded in six-well plates overnight and transfected with 100 nM siTMEM47#1, siTMEM47#2, or siNC (negative control). At 24 h post-transfection, the cells were treated with SVCV (MOI = 1). At 24 h post-stimulation, total RNAs were extracted to examine the transcriptional levels of tmem47. (F and G) Effects of TMEM47 RNAi on the SVCV/CyHV-2-induced IFN and other ISGs transcription. EPC or GiCB cells were seeded in six-well plates and transfected with 100 nM siNC, siTMEM47#1, or siTMEM47#2. At 24 h post-transfection, cells were treated with SVCV for 24 h or CyHV-2 for 48 h before qPCR analysis was performed. (H–K) EPC or GiCB cells seeded in 24-well plates overnight were transfected with 0.5 µg TMEM47-Myc or empty vector. At 24 h post-transfection, cells were infected with SVCV (MOI = 0.001) for 48 h or CyHV-2 (MOI = 0.001) for 72 h. Then, cells were fixed with 4% paraformaldehyde (PFA) and stained with 1% crystal violet (H and J). Culture supernatants from the cells infected with SVCV or CyHV-2 were collected, and the viral titer was measured according to the method of Reed and Muench (I and K). Data were expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from control (*P < 0.05). All experiments were repeated for at least three times with similar results.