Fig. 1.

NS3/4A cleaves IPS-1 to control the host response during HCV infection. (A) Huh7 cells were mock-infected or infected with JFH-1 HCV 2a at a multiplicity of infection of 3. At the indicated h after infection, cells were immunostained with HCV 2a patient serum (green) and anti-IRF-3 serum (red). Nuclei were visualized by staining the cells with DAPI (blue). Cells with nuclear IRF-3 (indicated by arrows) were counted and expressed as a percentage of the total cells in a field. The top left of A shows IRF-3 in Sendai virus-infected control cells. (B) Focus-forming units per ml of HCV within supernatant collected from cultures of Huh7 cells (white bars) or Huh7.5 cells (black bars) at 24 and 48 h after infection with JFH-1 virus. (C) Immunoblot of endogenous IPS-1, NS3, NS5A, and GAPDH in Huh7 cells that were mock-infected (lanes 1–4) or infected with the JFH-1 virus (lanes 5–8) for the time shown above each lane. Arrows mark the positions of full-length (FL) and cleaved (C) forms of IPS-1. (D) Immunoblot of endogenous IPS-1, NS3, and GAPDH in extracts from Huh7 (lane 1), Huh7-K2040 (lane 2), Huh7-HP (lane 3), and Huh7-JFHR (lane 4) cells and Huh7 cells that were mock-infected or infected with the JFH-1 virus (multiplicity of infection = 1) for 96 h (lanes 5 and 6, respectively). (E) Huh7 cells were cotransfected with constructs encoding the Myc-vector (−) or Myc-IPS-1 (+) and either the Flag-vector (lanes 1–2) or a Flag-tagged expression construct encoding WT NS3/4A protease from the HCV 1b Con1 clone (lanes 3–4), protease-deficient Con1 mutant (lanes 5–6), patient isolates (HCV 1a, lanes 7–8; HCV 1b, lanes 9–10; HCV 2b, lanes 11–12), or the JFH-1 clone (lanes 13–14). Expression of Myc-IPS-1, Flag-NS3, ISG56, and GAPDH is shown.