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

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

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

Identification and characterization of potential adaptive mutations.

<p>(<b>A</b>) Positions of conserved mutations found in the adapted virus on JFH1 open reading frame schematic diagram. The originally introduced amino acid changes F172C and P173S in Core and A4 MAb epitope in E1 are indicated in underlined gray type. Mutations identified at the end of the selection are indicated in black type. (<b>B</b>, <b>C</b>, <b>D</b>) Effect of the potential adaptive mutations on viral genome replication, infectious virus production and HCVcc assembly/secretion. HuH-7-RFP-NLS-IPS cells were transfected with JFH1-CS-A4-RLuc RNA (WT) or mutated HCV genomes (I599V, R1373Q, M1611T, S2364P, C2441S, R2523K, R1373Q/C2441S (DM for double mutant) or R1373Q/M1611T/C2441S (TM for triple mutant)). An assembly-deficient virus (ΔE1E2) and a replication-defective virus (GND) were used as controls. (<b>B</b>) Replication was assessed at 4, 24 and 48 h by measuring <i>Renilla</i> Luciferase activities in transfected cells. Results are expressed as relative light units (RLU) normalized at 4 h and are reported as the means ± S.D. of two independent experiments. (<b>C</b>) The supernatant of transfected cells were recovered at 24 and 48 h and incubated for 3 h with naive HuH-7-RFP-NLS-IPS cells. Luciferase assays were performed on infected cells at 72 h post-infection. Results are expressed as RLU and are reported as the means ± S.D. of two independent experiments. (<b>D</b>) HCV core release was quantified in the supernatants recovered 48 h post-transfection. Results are expressed as Core fmol/L and are reported as the means ± S.D. of two independent experiments.</p

Viral entry of cell culture adapted HCV.

<p>(<b>A</b>, <b>B</b>, <b>C</b>) Neutralization of cell culture adapted HCV with 3/11 and JS-81 MAbs. HuH-7-RFP-NLS-IPS cells were infected with i0 or i24 in the absence (Mock) or the presence of 3/11 anti-E2 or JS-81 anti-CD81 MAbs, at the indicated concentration. (<b>A</b>) Images taken 48 h after infection with i24 are representative of three independent experiments. (<b>B</b>, <b>C</b>) Intracellular HCV RNA was quantified 48 h after infection. Results are expressed as percentages of infectivity relative to infectivity in the absence of antibodies and are reported as the means ± S.D. of two independent experiments. (<b>D</b>) Cell-to-cell transmission of cell culture adapted HCV. Naive HuH-7-RFP-NLS-IPS cells (acceptor cells) were seeded with HuH-7-EGFP-IPS cells, infected with either i0 or i24 (donor cells). Cultures were treated with 50 µg/mL of the 3/11 anti-E2 neutralizing MAb to prevent cell-free infection. The results are expressed as the mean number of HCV infected acceptor cells/focus ± S.D., determined in 140 separate foci, 24 h post-seeding.</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

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