FIG. 3.

GST-CD81 inhibition of HCVpp infectivity. (A) Human and mouse GST-CD81 LEL (hLEL and mLEL, respectively) proteins were either added to Huh-7.5 cells (“cells”) and incubated for 1 h at 37°C before cells were washed and pseudoparticles added, or CD81 LEL was mixed with pseudoparticles (“virus”) for 1 h at 37°C before adding the mixture to cells. In both cases the final concentration of CD81 LEL was 10 μg/ml. White bars represent HCVpp-H77; shaded bars represent MLVpp. Three days after infection, cells were lysed and luciferase activity determined. Cells infected with no-Env control virus gave a mean luciferase reading of 2,134 ± 308 (data not shown). (B and C) A high-titer infectious preparation of HCVpp-H77 was incubated with no inhibitor or with 5 μg/ml of human CD81 LEL and C1 anti-E2 MAb for 1 h at 37°C before infection. (B) Aliquots of the untreated and treated virus mixtures were tested for infectivity on Huh-7.5 cells. (C) The remaining virus was diluted 60-fold (to reduce the CD81 LEL and C1 to a biologically inactive concentration) and tested for infectivity in the presence and absence of additional human CD81 LEL and C1 MAb at a final concentration of 5 μg/ml. Three days after infection, cells were lysed and luciferase activity determined. Cells infected with no-Env control virus gave mean luciferase readings of 5,006 ± 715 and 856 ± 92 at the two respective dilutions (data not shown). (D and E) Human and AGM CD81 LEL inhibition of HCVpp-H77 infection of HepG2 cells expressing either human (D) or AGM (E) CD81. All infections were performed in triplicate, and the data are representative of two independent experiments (mean ± standard deviation). RLU, relative light units.