Hepatitis C Virus Resistance to Carbohydrate-Binding Agents

Izquierdo, Laure; Oliveira, Catarina; Fournier, Carole; Descamps, Véronique; Morel, Virginie; Dubuisson, Jean; Brochot, Etienne; Francois, Catherine; Castelain, Sandrine; Duverlie, Gilles; Helle, Francois

We used a plasmid encoding JFH1-CS-A4 genome, a modified version of the full-length JFH1 strain (genotype 2a; GenBank access number AB237837; kindly provided by T. Wakita, National Institute of Infectious Diseases, Tokyo, Japan), which contains mutations leading to amino acids changes F172C and P173S at the C-terminus of the Core protein that increase the viral titers [23] . In this construct, the sequence encoding residues 196 TSSSYMVTNDC at the N-terminal region of E1 has also been modified to reconstitute the A4 epitope (SSGLYHVTNDC) [22] , as previously described [24] . Additionally, to characterize the mutations by reverse genetics, we used a plasmid encoding the JFH1-CS-A4-Rluc-DM genome (referred to as wild-type (WT) in this study), which contains a Renilla Luciferase reporter gene and two adaptive mutations (R1373Q and C2441S in NS3 and NS5A, respectively), as described previously [17] . These plasmids were used to produce HCV RNA by in vitro transcription which were electroporated into HuH-7-RFP-NLS-IPS cells, as previously described [17] . Supernatants of electroporated cells were recovered and filtered through a 0.45-μm-poresized membrane, aliquoted and stored until use at -80°C.