FIG. 3.

Role of specific CD81 LEL residues in HCVpp entry and sE2 binding. (A) HCVpp entry was tested in HepG2 cells transfected with pcDNA3.1, human wild-type CD81, murine wild-type CD81 (all in white bars), and human CD81 mutants, as indicated along the x axis. Human residues were replaced by alanine (black bars) or by corresponding murine (shaded bars) or rat (hatched bars) CD81 residues. HCVpp entry was calculated as a percentage of the entry level into cells expressing human wild-type CD81. The expression of CD81 mutants was quantified by flow cytometry after labeling of cells with JS81 (or 1D6 for the A164T mutant) and was expressed as a percentage of human wild-type CD81 expression. The percentage of HCVpp entry was further normalized for CD81 expression. Values are means of three independent experiments ± SD. (B) Binding of sE2 to CD81 mutants with significantly decreased HCVpp entry was quantified by flow cytometry. HepG2 cells were transfected with pcDNA3.1, human wild-type CD81, murine CD81, and CD81 mutants. Transfected cells were incubated with sE2, followed by labeling with H53 and a PE-conjugated anti-mouse IgG. Binding was analyzed by flow cytometry and expressed as a percentage of sE2 binding to HepG2 cells expressing human wild-type CD81. The percentage of sE2 binding was further normalized for mutant expression levels. Values are averages of three independent experiments ± SD.