, 2010 and Mondal et al., 2009). In 2005, an HCV gt2 infectious clone was described supporting the production of infectious HCV particles in cell culture (HCVcc), enabling for the first time the investigation of the full viral life cycle (Lindenbach et al., 2005, Wakita et al., 2005 and Zhong et al., 2005). An infectious cell culture system for full length HCV gt1 was reported which is the most prevalent genotype worldwide (Li et al., 2012 and Yi et al., 2006), however, screening involving cell-culture adapted HCV have only been performed for gt2 and gt1/2 chimeric viruses (Chockalingam et al., 2010,

Gastaminza et al., 2010, Gentzsch et al., 2011 and Wichroski et al., 2012). SB203580 cell line In this context, by combining the gt1 replicon with the infectious HCV gt2 cell

culture system our goal was to develop a high-throughput phenotypic assay to identify cross-genotype antivirals with a novel selleck chemicals llc mechanism of action. Our devised strategy allows multiparameter data acquisition from a single well by a phenotypic approach by combining (i) the identification of novel HCV inhibitors with cross-genotypic activity, (ii) indication of the targeted stage of the virus life cycle, and (iii) early assessment of compound induced cytotoxicity. Taking advantage of the observation that the mitochondrial antiviral signaling protein (MAVS/IPS-1), located in the outer mitochondrial membrane, is a cellular substrate for the HCV NS3-4A protease, Jones et al. developed a cell-based fluorescent reporter system allowing sensitive detection of HCV-infection in live cells (Jones et al., 2010 and Loo et al., 2006). Overexpression of a fusion protein consisting of the membrane anchored C-terminal IPS-1 domain linked to a nuclear localization signal (NLS) and red fluorescent protein (RFP) (Fig. 1A), enables monitoring HCV infection events by measuring the translocation of cytoplasmic localized RFP into nucleus upon by NS3-4A protease mediated cleavage between RFP-NLS and IPS-1 (Fig. 1A and B). To establish the phenotypic multiplex assay, Huh-7.5 derived RFP-NLS-IPS reporter cells were mixed at 1:2 ratio with Huh-7 gt1b replicon

cells, expressing an HCV NS5A-GFP fusion protein as a marker for viral replication (Moradpour et al., 2004), and co-plated into one well (Fig. 1A). The experimental protocol can be briefly described as follows: 2,400 cells L-gulonolactone oxidase per well were plated into 384-well assay plates, at 24 h post-plating compounds were added and after a 2 h incubation period at 37 °C, cells were inoculated with Jc1 (Lindenbach et al., 2006 and Pietschmann et al., 2006), a reporter-free gt2a virus at a multiplicity of infection of 2 (Fig. 1A). At 72 h post-infection, plates were fixed with 2% paraformaldehyde, cell nuclei were stained with 10 μg/mL Hoechst-33342 and images were taken with an automated confocal microscope (ImageXpress Ultra, Molecular Device) at a magnification of 20×.