Fig 2.

Adaptive coding mutations are indispensable for enhanced replication fitness. (A and B) Huh-7 cells were infected at a MOI of 500 HCV GE/per cell with the indicated molecular clones. Replication fitness was analyzed by (A) RT-qPCR for intracellular viral RNA in total cellular RNA and (B) by virus titer determination of cell culture supernatant at the indicated time points. Statistical significance was calculated using two-way ANOVA with Dunnett’s multiple comparisons test with single pooled variance and significance levels of P < 0.05 = *, P < 0.01 = **, P < 0.001 = ***, P < 0.0001 = ****. (C and D) Huh-7.5 and Huh-7.5 miR-122 deficient cells were infected with 140 HCV GE/cell of the indicated p100 consensus virus variants. At 72 h.p.i., cell culture supernatant and total cell lysates were collected and analyzed for (C) intracellular viral RNA and (D) released infectivity determined by a limiting dilution assay. Numbers indicate the fold-difference of samples collected from Huh-7.5 miR-122 deficient cells compared to samples from the parental cell line. (E and F) Huh-7 cells were infected with 340 HCV GE/cell of p100pop or p100 consensus virus harboring adaptive coding mutation without (p100NGS_coding) or with (p100NGS_coding_FLAG_PS) additional FLAG epitope and PS. Viral fitness was assessed by (E) RT-qPCR for intracellular viral RNA and (F) virus titer determination from cell lysates and cell culture supernatant collected at indicated time points. Numbers indicate fold-difference in viral fitness. Means and individual values in this figure are displayed for n = 3–4 experiments. Dashed lines represent the LOD of the performed limiting dilution assays.