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.