Figure 3.

TRIM22 has anti-viral activity against HCV. (a) Western blot analysis of TRIM22 expression with anti-TRIM22 antibody after transfection of cells with FLAG-TRIM22 (0.5 µg/well, 24-well plate). Endogenous TRIM22 is shown as a positive control in the lane labeled ‘IFNα' in Huh-7 cells that were treated with IFNα (50 IU/ml) for 24 h and collected for western blotting. (b, c) A marked decrease in the virus replicon levels was observed in TRIM22-over-expressing Huh-7-con1-replicon cells 48–96 h after infection. Huh-7-con1-replicon cells were transfected with GFP, TRIM22 and the anti-viral ISG and IRF1 plasmids. Samples were collected at 48 h, 72 h and 96 h after transfection. Real-time RT-PCR and western blotting were performed as described in the section on ‘Materials and methods'. (d) The knockdown efficiency of the siRNAs was determined by western blotting. Huh-7 cells were transfected with siRNA or TRIM22 (siRNA: 50 nmol per well/24-well plate, TRIM22: 100 ng per well/24-well plate), and cells were collected for western blotting 24 h after transfection. (e, f) Knockdown of TRIM22 diminished the anti-viral effects of IFNα. Huh-7 cells were transfected with siRNA (50 nmol per well/24-well plate). After 24 h, the transfected cells were infected with JFH1 (MOI=0.1). At 24 h after infection, the cells were treated with IFNα (50 IU/ml) for an additional 48 h. Samples were collected for real-time PCR assays and western blotting. *P<0.05 as determined by Student's t-test. ***P<0.001 as determined by Student's t-test. HCV, hepatitis C virus; IFN, interferon; IRF, IFN-regulatory factor; ISG, interferon-stimulated gene; PBMC, peripheral blood mononuclear cell; TRIM, tripartite motif.