Figure 1
A cell-based dual-reporter system for monitoring HCV infections.
(a) The rationale of the NIrD system. The sensor module consists of rtTA-MAVS(C) fusion proteins, which are constantly produced and localised to the mitochondria in cells. The amplifier module is an expression cartridge integrated into the chromosome that is composed of two reporter genes driven by the tight-TRE promoter. Upon HCV infection, the virally produced NS3-4A cleaves its recognition sequence in MAVS(C), releasing the free-formed rtTA into the nucleus. The tight-TRE promoter is activated by rtTA and Dox (2 μg/ml), resulting in the production of delta-TK-2A-mCherry proteins. After self-cleavage of the 2A peptide, mCherry shows red fluorescence and delta-TK phosphorylates GCV (2 μg/ml) to cause cell death. (b) mCherry signal of the NIrD system in response to an HCVcc infection. The Huh7.5(NIrD) system was infected by HCVcc for 72 h. HCVcc-transduced (+) or -untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a microscope. The fluorescence signals were superimposed onto white light images. Scale bar, 200 μm. (c) The death signal of the NIrD system in response to an HCVcc infection in the presence of GCV (2 μg/ml). Huh7.5(NIrD) cells were infected with HCVcc for 120 h. The transduced (+) or untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a light microscope. Scale bar, 200 μm. (d) Fluorescence microscopy of HCVcc core protein (Alexa Fluor 488) and mCherry signal upon HCVcc infection (72 h) in Huh7.5(NIrD) cells. Scale bar, 30 μm. (e) Immunoblotting analysis of Huh7.5(NIrD) cells infected with or without HCVcc in the presence or absence of Dox (2 μg/ml). HCVcc was detected by an antibody specifically targeting the viral core protein and β-tubulin was used as the loading control. (f) Dosage effects of VX-950 on the live-cell imaging of HCVcc-infected Huh7.5(NIrD) cells. Huh7.5(NIrD) cells were infected with HCVcc plus Dox (2 μg/ml) together with serially increasing dosages of VX-950 (Telaprevir). Fluorescence microscopic images were taken 72 h following the HCVcc infection. Scale bar, 200 μm.
