Huh-7 Cells Transfection with HCV NS3/5B

Huh-7 cells were inoculated at MOI 10 with a recombinant vaccinia virus expressing the T7 phage RNA polymerase (VacT7) [104 (link)]. Two hours later, cells were transfected with the plasmid pTM-NS3/5B [16 (link), 58 (link)] (kindly provided by Dr. Lohmann; U. of Heidelberg) and Lipofectamine 2000 (ThermoFisher-Massachussets, USA) following the manufacturer´s recommendations in terms of total DNA per well (typically 4μg per 35mm dishes with 7.5 X 10⁵ cells/well) and 50% of the recommended lipofectamine:DNA ratio. Transfected cells were cultured in the presence of the DNA replication inhibitor cytosine β-D-arabinofuranoside (AraC; Sigma-Aldrich) for 16 hours to prevent VacT7 replication [105 (link)]. When indicated, media was also supplemented with 100nM daclatasvir (DCTV). Total cell extracts were used to determine viral protein accumulation by Western-blot using anti-NS3 antibody (clone 2E3; Biofront) and β-actin (Abcam; ab8226) as loading control.