Fig. 2.

RIG-I signals IRF-3 to control HCV replication and is regulated by NS3/4A. (A and B) Control Huh7 cells were transfected with vector only. Huh7-A7 cells harboring the A7 HCV replicon (A7) or Huh7-HP cells harboring the HP HCV replicon (HP) were transfected with 1 μg of plasmid DNA encoding vector alone, IRF-3-5D or IRF-3-ΔNor1 μgor2 μg of N-RIG expression plasmid. Cells were harvested 48 h posttransfection, and extracts were subjected to Northern blot analysis (A) and immunoblot analysis (B) using specific DNA or antibody probes, respectively. (C) UNS3/4A cells, cultured to suppress (-NS3/4A) or induce (+NS3/4A) NS3/4A expression, were transfected with the N-RIG expression construct, and 24 h later were subjected to dual immunostaining for ectopic N-RIG and endogenous IRF-3. Panels show N-RIG, IRF-3, and DAPI-stained nuclei. (D) UNS3/4A cells cultured to suppress or induce NS3/4A expression were infected with NDV as shown (Left) or were transfected with N-RIG expression plasmid (Right). After 24 h, cells were harvested, and protein extracts were separated on nondenaturing gels and subjected to immunoblot analysis to define the dimer, monomer, and phosphoserine 386 (Ser-386-P) isoforms of IRF-3. (Bottom) NS3 levels derived by standard denaturing gel immunoblot analysis.