EWI-2wint inhibits the infection of both HCVcc and HCVpp.

<p>A, Cellular populations (pcDNA3.1, HAEWI-2FLAG and EWI-2FLAG^fur POP) and individual cellular clones (EWI-2FLAG^fur A1, A6, B14, C1) were infected with HCVcc expressing <i>Renilla</i> luciferase (R-Luc). In parallel, these cells were infected with HCVpp 1a, HCVpp 2a or VSVpp expressing firefly luciferase (F-Luc) (B), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001866#pone-0001866-g006" target="_blank">Figure 6</a>. At 2 days post-infection, cells were lysed and processed to measure the luciferase activity. The luciferase activities were normalized for protein concentration in each cell lysate. The results are presented as relative percentages to HCVcc (A) and HCVpp (B) infectivity on Huh-7 cells. HCVpp infections (B) were also normalized to VSVpp infections on Huh-7 cells. Results are reported as the mean±S.D. of three independent experiments.</p

The interaction between HCV glycoproteins and CD81 is blocked when CD81 interacts with partner protein(s).

<p>A, HCV E1E2 heterodimers immobilized onto anti-E2 coated beads interacted with CD81 in all cells lysed in Triton X-100 (TX). However, the interaction of HCV-E1E2 with CD81 was blocked in Daudi and Ramos cells lysed in Brij (Bj). Maintenance of CD81 with other tetraspanins (T) and partners (P) is disrupted by the indicated lysis conditions, as diagrammed. Anti-CD81 and irrelevant (Cont) mAbs were used in immunoprecipitations as controls. Precipitation of CD81 was revealed by western blotting with the anti-CD81 5A6 mAb. CD81-LEL corresponds to the large extracellular loop of CD81 fused to the glutathione-S transferase. B, After cell surface biotinylation, the indicated cell lines were lysed with Bj/EDTA, immunoprecipitated with 5A6 mAb and the proteins revealed by Western blotting with HRP-conjugated streptavidin. The values on the right are molecular sizes in kilodaltons. C, EWI-2wint production is directly connected to EWI-2 expression. Daudi cells interfered with negative siRNA or EWI-2 siRNA were biotinylated, lysed in Bj/EDTA, immunoprecipitated with the anti-CD81 5A6 mAb or an anti-EWI-2 mAb (8A12) and blotted sequentially with HRP-conjugated streptavidin and 5A6 mAb.</p

Mass spectrometric analyses and N-terminal sequencing of EWI-2 and EWI-2wint proteins

<p>Tryp: trypsine; Glu-C: V8 endoproteinase Glu C</p

Alignment of EWI-2 sequence and the deduced EWI-2wint sequence.

<p>Asterisk indicates the position at which an Arg (R) residue was introduced to make a furin cleavage site. Underlined amino acids correspond to the signal peptide of EWI-2. The four Ig domains are colored. Amino acid residues corresponding to the putative transmembrane domain are in italics and those corresponding to the cytoplasmic tail are bolded.</p

EWI-2wint inhibits the entry stage of HCV life cycle by reducing the interaction between E1E2 and CD81.

<p>A, EWI-2wint does not interfere with HCV replication. The indicated Huh-7 cell lines were transfected with the full-length JFH1 genome, and expression of the core antigen was evaluated at 40 h post-transfection. The displayed fields contained similar numbers of cells. B, NS3 expression in JFH-1 transfected cells. Cells were stained by using an anti-NS3 mAb (486-D39) and secondary antibody conjugated with PE. Cont corresponds to untransfected Huh-7 cells. C, Huh-7 cell lines were incubated for 3 h with virus pseudotyped with HCV envelope glycoprotein (HCVpp) or VSV G envelope protein (VSVpp). HCVpp were generated with envelope proteins from 1a (HCVpp 1a) or 2a (HCVpp 2a) genotype. The inoculum was then removed and the cells were further incubated. At 2 days post-inoculation, cells were lysed and processed to measure the luciferase activity. The luciferase activities were normalized for protein concentration in each cell lysate. The results are presented as relative percentages to HCVpp infectivity on Huh-7 cells. Results are reported as the mean±S.D. of three independent experiments. Pseudotyped particles produced in the absence of envelope proteins were used as controls. The mean fluorescence activity of such particles represented less than 2% of the activity measured for HCVpp. D, <i>In vitro</i> interaction of E1E2 heterodimers with CD81 from Huh-7/EWI-2FLAG^fur and Huh-7/pcDNA3.1. This assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001866#pone-0001866-g001" target="_blank">Figure 1</a>. The ratio [CD81 band intensity in E1E2 IP in Bj/EDTA buffer]/[CD81 band intensity in E1E2 IP in TX/EDTA buffer] was 0,27 and 0,84 in Huh-7/EWI-2FLAG^fur and Huh-7/pcDNA3.1, respectively.</p

EWI-2wint inhibits HCVcc infection.

<p>Cell clones were infected with JFH1 HCVcc and infectivity was analyzed by indirect immunofluorescence with an anti-C mAb (A) or Western blotting with an anti-E2 mAb (B). The fields displayed in (A) contained similar numbers of cells. C, Infection levels were measured by quantification with NIH Image 1.62 of the intensities of HCV E2 bands. The results are presented as percentages of HCVcc infection relative to the infection of Huh-7/pcDNA3.1 cells. Infection levels of three independent experiments are reported as the mean with standard deviation bars. D, CD81 expression on the surfaces of cells expressing or not EWI-2wint. Cells were stained by using an anti-CD81 mAb (5A6) and secondary antibody conjugated with PE. Cont corresponds to Huh-7 cells that were stained only with the secondary antibody.</p

EWI-2wint is a cleavage product of EWI-2.

<p>CHO cells were (co)-transfected with pcDNA3.1/CD81 and pcDNA3.1/EWI-2FLAG (A and D), with pcDNA3.1/wint-FLAG (B and E), or with pcDNA3.1/CD81 and pcDNA3.1/EWI-2FLAG^fur (C and F). EWI-2FLAG^fur corresponds to EWI-2FLAG in which a furin cleavage site has been engineered. After cell surface biotinylation, cells were lysed with Bj/EDTA (A–C) or TX/EDTA (D–F) and proteins were immunoprecipitated with the indicated mAbs. Proteins were revealed by Western blotting with HRP-conjugated streptavidin. Asterisks indicate additional cleavage products of EWI-2.</p

EWI-2wint expression in Huh-7 cells.

<p>After cell surface biotinylation, Huh-7 cells (A) stably expressing pcDNA3.1 (B), HAEWI-2FLAG (C) or EWI-2FLAG^fur (D) were lysed with Bj/EDTA and analyzed by immunoprecipitation with indicated mAbs. Proteins were revealed by Western blotting with HRP-conjugated streptavidin. The molecular weights of the prestained molecular ladder are indicated in KDa. The asterisk indicates an additional cleavage product of EWI-2. The band indicated by a light arrow likely corresponds to a dimer of CD81, and the band indicated by a full arrow to an unidentified EWI-2 associated protein.</p

Comparison of the crystal structure of E2c with the homology model.

<p>Structural superposition between E2c crystal structure from the PDB chain 4mwf_D (red) and the homology model (black) is illustrated using a ribbon representation. The crystal structure and homology model overlap in most of the regions, except the fragments where coordinates in the experimental structure are missing (red dashed lines).</p

The IC50 values obtained for the 23 ligands screened for their ability to inhibit HCVcc, HCVpp and RD114pp infection of Huh-7 cells.

<p>ND refers to molecules that were not assayed because the molecule was not specific for HCV (it inhibited RD114pp infection). *IC50 values for anti-CD81 antibody are in µg/ml.</p><p>The IC50 values obtained for the 23 ligands screened for their ability to inhibit HCVcc, HCVpp and RD114pp infection of Huh-7 cells.</p