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

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

CLDN6/R209Q and OCLN/P24A mutations do not affect the kinetics of HCV entry and cellular physiology.

<p>Huh-7 cells were transduced with CLDN6/wt or CLDN6/R209Q and OCLN/wt or OCLN/P24A either alone or in combination. (<b>A</b>) Forty-eight hours after the last transduction round, cells were infected with HCVcc for 2h, 1h30, 1h or 30min. After infection, cells were rinsed and fresh medium was added. At 30h post-infection, cells were lysed and <i>Gaussia</i>-luciferase activities were measured. Results were normalized to luciferase activities measured in cells expressing the wt forms of CLDN6 and OCLN. (<b>B</b>) Cells were infected with HCVcc at indicated m.o.i. in serum-free DMEM for 2 h. At 30 h post-infection, cells were lysed and <i>Gaussia</i>-luciferase activities were measured. Results were normalized to luciferase activities measured in cells expressing the wt forms of CLDN6 and OCLN. Results are presented as mean ± SD of two independent experiments. (<b>C</b>) Huh-7-Lunet-CD81-FLuc cells, which endogenously expressed the <i>Firefly</i>-luciferase reporter gene, were transduced with CLDN6/wt or CLDN6/R209Q and OCLN/wt or OCLN/P24A either alone or in combination. Forty-eight hours after the last transduction round, cells were infected with HCVcc expressing the <i>Gaussia</i>-luciferase reporter gene. At 30h post-infection, cells were lysed and luciferase activities were measured with the Dual-luciferase^® reporter assay system (Promega). <i>Gaussia</i>-luciferase activities were normalized to Huh-7-Lunet-CD81 cells <i>Firefly</i>-luciferase activities. Results were adjusted to 100% infection for cells expressing the wt forms of CLDN6 and OCLN. Results are presented as mean ± SD of at least three independent experiments.</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

CLDN6/R209Q and OCLN/P24A mutations do not affect HCV cell-to-cell transmission.

<p>Huh-7 cells were transduced with CLDN6/wt or CLDN6/R209Q mutant and OCLN/wt or OCLN/P24A mutant either alone or in combination. Transduced cells were cocultivated for 24 h with CMFDA-stained donor cells infected with (<b>A</b>) HCVcc 2a or (<b>B</b>) HCVcc 3a in the presence (cell-to-cell transmission) or in the absence (cell-free and cell-to-cell transmission) of the 3–11 neutralizing antibody, which prevents the cell-free transmission. Cells were stained with an anti-NS5 antibody and analyzed by flow cytometry. HCV-positive CMFDA-negative cell populations were considered as newly infected cells through cell-to-cell transmission. Results of cell-free (light grey) and cell-to-cell (black) transmission are presented as percentages relative to the total transmission.</p

CLDN6/R209Q mutation does not affect HCVpp entry.

<p>293T cells were transduced twice at 24h interval with CLDN6/wt or CLDN6/R209Q. Expression was analysed 48h after the last transduction round. (A) CLDN6/wt and CLDN6/R209Q cell surface expression was measured by flow cytometry using an anti-CLDN6 antibody, and compared to that of non-transduced 293T cells (293T Mock). Cells stained with secondary antibodies were used as negative controls (dashed line). (B) CLDN6/wt and CLDN6/R209Q expression in 293T cells was also analyzed by immunofluorescence with an anti-CLDN6 antibody. (<b>C</b>) Two days after the last transduction, 293T cells were infected with HCVpp1a, HCVpp2a or VSVpp expressing the <i>Firefly</i>-luciferase reporter gene. Non-transduced 293T cells (Mock) were infected in parallel. Results are presented in relative luciferase units (RLU). (D) 293T cells were transduced sequentially with a combination of lentivectors encoding SRB1, CD81 and/or OCLN in addition to CLDN6/wt or CLDN6/R209Q. Cells were next infected with HCVpp1a or VSVpp 48h after the last transduction round. Cells were lysed 72h post-infection and results were normalised with luciferase activities measured in cells infected with VSVpp. Values were adjusted to 100% infection for cells expressing CLDN6/wt. (<b>E</b>) 293T cells transduced to express SRB1, CD81 and OCLN in addition to CLDN6/wt or CLDN6/R209Q were infected with HCVpp1a or VSVpp. Cells were lysed 72h post-infection and results were normalised with luciferase activities measured in cells infected with VSVpp. Values were adjusted to 100% infection for cells expressing CLDN6/wt. Results are presented as mean ± SD of three independent experiments. *** means a <i>p</i> value below 0.001.</p

Functionality of CLDN6/R209Q and OCLN/P24A in HCV infection expressed in polarized cells or primary hepatocytes.

<p>(<b>A</b>) Two days after the last transduction round, HepG2-CD81-1SC3 cells were plated on transwells and polarization was induced. Once polarized, cells on the lower chamber of the transwell were infected with HCVcc2a. At 30h after infection, cells were lysed, total RNA was extracted and viral RNA was quantified by qRT-PCR. Results shown are from one experiment representative of three independent experiments. (<b>B</b>) Transduced PHH (Biopredic) were infected with cell culture adapted HCVcc2a [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142539#pone.0142539.ref036" target="_blank">36</a>]. Non-transduced PHH (Mock) and PHH transduced with a lentiviral vector expressing only the YFP were used as controls. Virus that had been inactivated at 60°C for 30 min (inactivated HCVcc) was used as another control. Infection levels were evaluated by quantifying HCV RNAs. Two independent experiments on PHH were performed. Results presented in B are the mean ± SD of triplicates done on PHH from one liver resection.</p

Schematic representation of mutated entry factors.

<p>Schematic representation of CLDN6 and OCLN tight junction proteins and position of R209Q and P24A mutations in CLDN6 and OCLN, respectively.</p

OCLN/P24A mutation does not affect HCVpp entry.

<p>786-O cells were transduced twice at 24h interval with OCLN/wt or OCLN/P24A. Expression was analysed 48h after the last transduction round. (A) OCLN/wt and OCLN/P24A expression in 786-O cells was estimated through the percentage of GFP positive cells by flow cytometry analysis. (B) OCLN/wt and OCLN/P24A export at the cell surface in transduced 786-O cells was analysed by microscopy. (<b>C</b>) Two days after the last transduction, 786-O cells were infected with HCVpp1a, HCVpp2a or VSVpp expressing the <i>Firefly</i>-luciferase reporter gene. Non-transduced 786-O cells (Mock) were infected in parallel. Results are presented in relative luciferase units (RLU). (<b>D</b>) 786-O cells were transduced sequentially with a combination of lentivectors encoding SRB1, CD81 and CLDN1 in addition to OCLN/wt or OCLN/P24A. Cells were infected 48h after the last transduction round with HCVpp1a or VSVpp. Cells were lysed 72h post-infection and results were normalised with luciferase activities measured in cells infected with VSVpp. Values were adjusted to 100% infection for cells expressing OCLN/wt. (<b>E</b>) 786-O cells transduced to express SRB1, CD81 and CLDN1 in addition to OCLN/wt or OCLN/P24A were infected with HCVpp1a or VSVpp. Cells were lysed 72h post-infection and results were normalised with luciferase activities measured in cells infected with VSVpp. Values were adjusted to 100% infection for cells expressing OCLN/wt. Results are presented as mean ± SD of four independent experiments. *** means a <i>p</i> value below 0.001.</p