Schedule of drug-escalating selection of GNA, CV-N, ConA and GRFT resistant HCV strains as a function of time.

<p>HuH-7-RFP-NLS-IPS cells were infected in the presence of GNA (<b>A</b>), CV-N (<b>B</b>), ConA (<b>C</b>) or GRFT (<b>D</b>). Infected cells were subcultured every three to four days in the presence of the lectins. When 100% cells were infected (indicated by arrows), supernatants were recovered and used to infect naive cells in the presence of the lectins. The concentrations of each lectin were increased in a stepwise manner as indicated. Stars indicate the time points when the virus isolates were recovered and sequenced.</p

Conservation of the mutated amino acids.

<p>Conservation of the mutated amino acids.</p

Effect of the E1E2p7 mutations on sensitivity to inhibition by GNA, CV-N, ConA and GRFT.

<p>Inhibition assays were performed by incubating WT or mutant HCVcc with various concentrations of GNA (<b>A</b>), CV-N (<b>B</b>), ConA (<b>C</b>) or GRFT (<b>D</b>). After a 1 h incubation at 37°C, mixes were put into contact with target cells for 4 h. Luciferase assays were performed on infected cells at 72 h post-infection. Results are expressed as percentages of infectivity compared to infection in absence of inhibitory protein and are reported as the means ± S.D. of at least three independent experiments.</p

Effect of the E1E2p7 mutations on E1E2 glycoprotein expression.

<p>HuH-7-RFP-NLS-IPS cells were transfected with WT or mutated HCV genomes. An assembly-deficient virus (ΔE1E2) was used as control. Expression of E1E2 viral glycoproteins and actin was analyzed in cell lysates 72h post-electroporation by western blotting with specific MAbs (A4 [anti-E1], 3/11 [anti-E2] and C4 [anti-actin]).</p

Effect of the E1E2p7 mutations on sensitivity to inhibition by MAb 3/11 and CD81-LEL.

<p>Inhibition assays were performed by incubating WT or mutant HCVcc with various concentrations of MAb 3/11 (<b>A</b>) or CD81-LEL (<b>B</b>). After a 1 h incubation at 37°C, mixes were put into contact with target cells for 4 h. Luciferase assays were performed on infected cells at 72 h post-infection. Results are expressed as percentages of infectivity compared to infection in absence of inhibitory protein and are reported as the means ± S.D. of at least three independent experiments.</p

Increase of HCV titers after successive infections.

<p>HuH-7 cells were electroporated in the presence of JFH1-CS-A4 RNA. Ten days later, the supernatant of electroporated cells was recovered (denoted supernatant i0) and used to perform successive infections in HuH-7-RFP-NLS-IPS. Each time the cells were 100% infected, the supernatant was recovered (supernatants recovered after “n” infection, denoted i1 to i24) and used to infect naive HuH-7-RFP-NLS-IPS cells. (<b>A</b>, <b>B</b>) The amount of HCV RNA (<b>A</b>) and Core protein (<b>B</b>) were quantified in these supernatants by RT-qPCR and fully automated chemiluminescent microparticle immunoassay, respectively. Results are expressed as HCV RNA copies/mL and fmol/L of HCV Core protein, respectively, and are reported as the mean ± S.D. of duplicate and triplicate measurements, respectively. (<b>C</b>) Viral titers were determined by ffu assay for i0, i6, i9, i12 and i24. Results are expressed as ffu/mL and are reported as the mean ± S.D. of three independent experiments. (<b>D</b>) HuH-7-RFP-NLS-IPS cells were inoculated with the different supernatants at low MOI. Foci of infected cells, identified by translocation of the cleavage product RFP-NLS to the nucleus, were visualized at 24 and 48 h. Images are representative of three independent experiments.</p

Cytopathic effects induced by cell culture adapted HCV.

<p>HuH-7-RFP-NLS-IPS cells were infected with i24 at different MOIs. Non-diluted virus that had been inactivated at 60°C for 30 min was used as control (ctrl). Infected cell viability was evaluated 3 days after infection. The results are expressed as percentages of viability compared to non-infected cells and are reported as means ± S.D. of three independent experiments.</p

Infection of PHHs with cell culture adapted HCV.

<p>PHHs from one representative donor were inoculated for 6 h with non-adapted HCV (i0; MOI = 0.01 HuH-7 infectious units per cell) or i24 (MOI = 1000 HuH-7 infectious units per cell), in the presence or absence of 2′CMC (10 µM) or py6 (500 nM). After inoculation, cells were washed three times with PBS and new media containing the drugs were added and replaced every day. (<b>A</b>) Infection of PHHs that had previously been transduced with lentivirus expressing RFP-NLS-IPS, was visualized 48 h post-infection by translocation of the cleavage product RFP-NLS to the nucleus (“Infection” panel). The supernatants of inoculated cells were recovered 48 h post-infection, centrifuged and used to inoculate naive HuH-7-RFP-NLS-IPS in the absence or presence of py6 (“Re-infection” and “Re-infection with py6” panels, respectively) to check the production of progeny virus. Infected HuH-7 cells were visualized 48 h post-infection. (<b>B</b>) Intracellular HCV RNA was quantified by RT-qPCR, after inoculation of non-transduced PHHs. Results are expressed as means ± S.D. of duplicates. (<b>C</b>) Expression of the viral proteins E1 and E2 was analyzed 48 h post-infection in cell lysates by Western blotting using specific MAbs (A4 [anti-E1], 3/11 [anti-E2]<b>,</b> and C4 [anti-β-actin]). HuH-7 cells infected in the same conditions were used as control. (<b>D</b>, <b>E</b>, <b>F</b>) IFN-β, IL-28A/B and IL-29 expression in infected PHHs was determined in duplicate by RT-qPCR. The results are normalized to GAPDH endogenous control and presented as fold-increase over pre-infection levels, using the ΔΔCt method.</p

Infection of hepatoma cell lines with cell culture adapted HCV.

<p>(<b>A</b>) HuH-7, HepG2-CD81, Hep3B, PLC/PRF/5, SNU-182, SNU-398, SNU-449, Cos-7 and Caco-2 cells transduced with lentivirus expressing RFP-NLS-IPS were mock-infected (left) or inoculated with i24 (middle) (MOI = 10000 HuH-7 infectious units per cell). Infected cells, identified by translocation of the cleavage product RFP-NLS to the nucleus, were visualized 48 h post-infection. The supernatants of inoculated cells were recovered 72 h post-infection, centrifuged and used to inoculate naive HuH-7-RFP-NLS-IPS to check the production of progeny virus (right). Images are representative of three independent experiments. (<b>B</b>) The permissivity of HuH-7, HepG2-CD81, Hep3B and PLC/PRF/5 cells to the cell culture adapted virus was determined by TCID₅₀ assay. The results are expressed as TCID₅₀/mL ± S.D. calculated on 8 wells. (<b>C</b>) miR-122 expression was determined by RT-qPCR in HuH-7, HepG2-CD81, Hep3B, PLC/PRF/5, SNU-182, SNU-389, SNU-449, Cos-7 and Caco-2 cells. The results, which are representative of four independent experiments, are expressed as relative miR-122 expression using the ΔΔCt method with RNU6B as endogenous control and HuH-7 cells as calibrator.</p

Profiles of density of HCV produced in different hepatoma cell lines.

<p>HuH-7 (<b>A</b>), Hep3B (<b>B</b>), HepG2-CD81 (<b>C</b>) and PLC/PRF/5 (<b>D</b>) were electroporated with <i>in vitro</i> transcribed RNA of the JFH1-CS-A4-RLuc genome containing mutations R1373Q/C2441S. The supernatants of each electroporated cell lines were recovered six days post-electroporation and overlaid on 10 to 50% (weight/volume) iodixanol gradients. After a 24 h ultracentrifugation, sixteen fractions were collected and analyzed for HCV RNA quantity and infectivity on naive HuH-7 cells (assessed by measuring <i>Renilla</i> Luciferase activities). The results are expressed as percentages of total infectivity or HCV RNA and are reported as means of two independent experiments.</p