Characterization of HuH-7 cells encapsulated in Ca-alg beads at day 3 in culture.

<p>(A) Cell distribution in the Ca-alg beads. Cell viability was assessed using propidium iodide and acridine orange which stained dead and live cells, respectively. Scale bar 250 µm (B) The matrix porosity of Ca-alg beads was visualized by cryoscanning electron microscopy. Scale bar 3.75 µm.</p

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

Protective property of Ca-alg beads against various enveloped and non-enveloped viruses.

<p>Following the HuH-7 encapsulation stage, 600 µL of beads were transferred to tissue culture 6-well plates with 1 mL of complete DMEM medium added on 3D moving plates. After 4 h of Sindbis virus, HSV-1 and Poliovirus type 1 infection, the medium was removed and replaced with fresh complete DMEM medium. The Ca-alg beads devoid of cells were used as controls. The supernatants of the cultured cells were recovered at 48 h and incubated for 4 h with HuH-7 cell 2D cultures. Infectious particle production was assessed by TCID₅₀ titration using microscopy imaging analysis. **<i>P</i><0.001.</p

Absence of production of new HCVcc particles by previously infected and encapsulated cells.

<p>(A) Encapsulated cell cultures were established using JFH1-RLuc virus-infected HuH-7 cells or non-infected cells within Ca-alg beads. Following the cell encapsulation stage, 400 µL of beads were transferred in tissue culture 6-well plates with 1 mL of complete DMEM medium added on 3D moving plates. (B) Foci of infected (a) or non-infected (b) HuH-7-RFP-NLS-IPS cells identified by translocation of the cleavage product RFP-NLS from cytoplasm to nucleus, were visualized at 6 days post-encapsulation by fluorescence microscope. Images are representative of three independent experiments. Nuclei were stained by DAPI. Scale bar 20 µm. The supernatants of the bead culture cells were collected at day 6 and incubated for 4 h with HuH-7 cell 2D cultures. (C) The amount of HCV RNA was quantified in the bead culture supernatants by RT-qPCR. Results are expressed as HCV RNA IU/mL and are reported as the mean ± S.D. of triplicate measurements. (D) Infectious particle production was assessed luciferase assay on infected cells at 72 h post-infection. Results are expressed as RLU and are reported as the means ± S.D. of three independent experiments. (E) Infectious particle production was also assessed by measuring <i>Renilla</i> luciferase activities after bead degelification to free previously infected cells and plating them. Results are expressed as relative light units (RLU) and are reported as the means ± S.D. of three independent experiments. **<i>P</i><0.001, ***<i>P</i><0.0001.</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

Protective property of Ca-alg beads against HCV infection is dependent on bead/virus volume ratio concentration and time of incubation.

<p>Ca-alg beads devoid of cells were produced. (A) 1200 µL of Ca-alg beads were transferred in tissue culture 6-well plates with 1 mL of complete DMEM medium added on 3D moving plates. After three incubation times (0.5, 2 and 20 h) at room temperature of JHF1-RLuc virus with a Ca-alg bead/virus volume ratio of 4/1, the supernatant was recovered and incubated for 4 h with HuH-7 cell 2D cultures. The detection of infectious particles was assessed by luciferase assay on infected cells at 72 h post-infection. Results are expressed as RLU and are reported as the means ± S.D. of three independent experiments. (B) The same experiment was performed with different Ca-alg bead/virus volume ratios (0/1 to 8/1) for 20 h. As previously described, the supernatant was recovered and incubated for 4 h with HuH-7 cell 2D cultures. Luciferase assays were performed on the infected cells at 72 h post-infection. The Ca-alg beads without the JFH1-RLuc virus were used as controls (Ctrl). Results are expressed as RLU and are reported as the means ± S.D. of three independent experiments. (C) After 20 h post-incubation of JFH1 virus in a Ca-alg bead/virus volume ratio of 4/1, the Ca-alg beads were washed, degelified by lyase treatment. Viral titers of the supernatants were determined by FFU assay. Results are converted into a percentage of infectivity. (D) Under the same incubation conditions as C), the amount of HCV RNA was also quantified in the supernatants (condition without beads) and from the Ca-alg bead products after lyase treatment (condition with beads) by RT-qPCR. Results are expressed as HCV RNA IU/mL and are reported as the mean ± S.D. of triplicate measurements. *<i>P</i><0.05, **<i>P</i><0.001, ***<i>P</i><0.0001.</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

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