Multiple cell lines were employed in this study, including human hepatoma cell lines (Huh7 and PLC/PRF/5), a human embryonic kidney cell line (HEK293), a human primary glioblastoma cell line (U87), and a human fetal lung fibroblast cell line (MRC5). Huh7 and U87 cells were kindly provided by Professor Bart Haagmans from the Department of Viroscience, Erasmus Medical Center. The human embryonic kidney 293 cell line, PLC/PRF/5 and MRC5 were originally obtained from ATCC (
http://www.atcc.org). These cells were cultured in Dulbecco’s modified Eagle medium (Lonza Biowhittaker, Verviers, Belgium) supplemented with 10% fetal bovine serum, 100 IU of penicillin per ml, and 100 μg of streptomycin per ml. For the full-length HEV model, a plasmid construct containing the full-length HEV genome (Kernow-C1 p6 clone; GenBank accession number JQ679013) was employed to generate HEV genomic RNA using an Ambion mMessage mMachine
in vitro RNA transcription Kit (Thermo Fisher Scientific Life Sciences) [
16]. Huh7, PLC/PRF/5, HEK293, U87 and MRC5 cells were electroporated with full-length HEV genomic RNA to generate consecutive HEV-infected cell models (Huh7-p6, PLC/PRF/5-p6, HEK293-p6, U87-p6 and MRC5-p6). To generate the subgenomic (p6-Luc) HEV model, a plasmid construct containing subgenomic HEV was used. This plasmid has an HEV sequence in which the 5’ portion of HEV ORF2 was replaced with the in-frame
Gaussia princeps luciferase reporter gene [
16]. Huh7, U87 and HEK293 cells were electroporated with HEV subgenomic RNA to generate HEV subgenomic models (Huh7-p6-Luc, U87-p6-Luc, and HEK293-p6-Luc). To normalize nonspecific effects of 2CMC on the luciferase signal, Huh7 cells stably expressing a non-secreted firefly luciferase under the control of the human phosphoglycerate kinase (PGK) promotor (PGK-Luc) were used [
18]. In addition, Huh7 cells harboring a subgenomic HCV bicistronic replicon (I389/NS3-3 V/LucUbiNeo-ET) (Huh7-HCV-Luc) were used as positive control of antiviral activity.