Figure 4.

Lipoprotein receptor SR-B1 and LDLr do not affect HCV replication, SR-B1 facilitates GP-driven cell entry, and both redundantly participate in HCV infection. HCV GP-mediated entry in lipoprotein-deficient cells (A). Huh-7.5 parental (black bars), SR-B1 (light orange bars), LDLr (grey bars), and DKOs (light blue bars) were transduced with NLuc-encoding GT1a H77 or GT2a J6 HCVpp, negative (NoEnvpp), and positive (MLVpp) controls for 6 h. Six hpt, lentiviral particles were removed and reporter expression measured 72 h after. Raw relative light units (RLU) values were normalized to MLVpp values of the same cell line to account for intercellular variation. NoEnvpp normalized values were subtracted to represent HCV GP-specific entry. HCV subgenomic replication in Huh-7.5 and lipoprotein receptor-deficient cells (B). Cells were transfected with a HCV GT2a replication-competent (WT) or deficient (ΔGDD) subgenomic genomes tagged with FLuc. RLU values were normalized to 4 h post-electroporation and to MTT values to account for initial RNA delivery and cell growth variabilities between cell lines. Graph represents the average of three independent experiments performed in triplicate +/− sem. HCVcc infection of lipoprotein-deficient and parental cells (C). Indicated cell lines were infected untagged GT2a Jc1 HCVcc. Virus inoculum was removed hpi. Twenty-four hpi cells were lysed and Intracellular HCV RNA copies determined with an X-tail based absolute RT-qPCR. Data are shown as violin plots with median (black line) and individual values (black dots). Data distribution is symbolized by the plots’ shape. A two-way ANOVA with Dunnett post hoc (α = 0.05, DF = 10) was used for statistical analysis of HCV entry and replication assays and a one-way ANOVA with Sidak’s post hoc for HCVcc infection assay (α = 0.05, DF = 25). p-value was calculated and shown as: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.