JFH1-AM120 HCV reporter viruses with gene inserts into NS5A; JFH1-AM120-Rluc and JFH1-AM120-EGFP.

<p>(Panel A) Schematics of the HCV JFH1-wt and JFH1-AM120 based reporter constructs. EGFP and <i>Renilla</i> luciferace (Rluc) genes were inserted in frame into a unique RsrII site located in the amino acid 397 codon of JFH1-wt and JFH1-AM120. (Panel B) <i>In vitro</i> transcribed RNAs were electroporated into Huh-7.5 cells. Transfected cells were passaged for six days, infectivity titers of culture supernatants were measured and viral titers are expressed as focus-forming units per milliliter (f.f.u./ml) (triplicates that were performed twice). The data are presented as mean ± standard deviation (n = 6). (Panel C) JFH1-AM120-EGFP virus compared to JFH1-wt-EGFP. Huh-7.5 cells were inoculated with supernatants collected at six days post-transfection. Cells were fixed at 48 h post-infection and infected cells were identified by fluorescence immunostaining and microscopy as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044965#pone-0044965-g003" target="_blank">Figure 3</a>. Nuclear DNA was stained with DAPI (blue). (Panel D) <i>Renilla</i> luciferase activity was measured in Huh-7.5 cells following infection with day six supernatants from cells transfected with JFH1-wt-Rluc and JFH1-AM120-Rluc RNA. Assays were done in triplicate and in two separate experiments. The data are shown as mean ± standard deviation (n = 6).