Primers used to sequence the SCARB1 gene.

<p>Primers used to sequence the SCARB1 gene.</p

Primers used to sequence the CLDN6, CLDN9 and OCLN genes.

<p>Primers used to sequence the CLDN6, CLDN9 and OCLN genes.</p

Schematic representation of of CLDN6, CLDN9, OCLN and SCARB1 genes.

<p>Exons are represented by numbered gray rectangles and introns or non coding regions by double blue lines. Positions of primers pairs used for direct genomic sequencing are shown. Reference sequences of the transcripts are NM_021195.4 (CLDN6), NM_020982.3 (CLDN9), NM_002538.3 (OCLN) and NM_005505.4 (SCARB1). The numbering starts at the first base of the initiation codon ATG, and stops at the first base of the termination codon of the corresponding transcripts. The sequences of corresponding primers are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142698#pone.0142698.t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142698#pone.0142698.t002" target="_blank">Table 2</a>. F: Forward primer, R: Reverse primer, LR: Long Range PCR primer. SNPs identified by direct genomic sequencing are indicated, in red SNPs specific of the case population HIV+ HCV-.</p

Variants of HCV entry factors found in HIV-infected, HCV-uninfected patients but not in HIV/HCV-coinfected controls.

<p>^a Database single nucleotide polymorphism. ^b 1000 Genomes database. ^c refSNP reference identification number of single nucleotide polymorphism. ^d Not attributed. ^e Minor allele frequency. ^f Intravenous drug users.</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

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

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

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 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

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