HCV E1E2 gp expression. Transfected 293-T cells were tested for expression of HCV E1 and E2 gps. Cells transfected with pNL4.3.Luc.R^-E^- and pE1E2 were tested for cell surface and total expression of E1 (mAb H111), E2 (mAbs CBH5 or H52), and PDI (SPA-891). Cells were fixed and analyzed for antigen expression with (B) or without (A) permeabilization. Regions were established based on irrelevant isotype-matched IgG staining of the cells.

HIV pseudotype infectivity. (A) HIV pseudotypes bearing strain H native E1 and E2 gps alone or together (H E1, H E2, or H E1E2), strain Con1 E1E2 gps (Con1 E1E2), E1 and E2 ectodomains fused to the transmembrane domain and cytoplasmic tail of VSV G (H E1E2-G), SIN E2 and E1 gps (H E1E2-SIN), HIV–SF162 gp160 (SF162), VSV-G, or no envelope (no env) were tested for their ability to infect Huh-7.5 (filled bars) or Hos.CD4.R5 cells (hatched bars). All virus stocks were normalized for p24 HIV core antigen and infected at 1 ng per well, with the exception of VSV G, which was used at 0.1 ng per well. All infections were performed in triplicate, and the mean luciferase activity (RLU) is shown. The coefficient of variance was <10% in all cases. (B) Increasing concentrations of pseudotypes bearing H E1E2 (▴), SF162 (•), and no envelope (□) were tested for infection of Huh-7.5 and Hos.CD4.R5 cells.

pH dependency of HIV–HCV pseudotype infectivity. Huh-7.5 cells were infected with HIV pseudotypes bearing amphotropic murine leukemia virus (A-MLV), VSV-G, H E1E2, and Con1 E1E2 in medium alone (open bars) or containing 10 mM ammonium chloride (filled bars) or 25 nM concanamycin A (hatched bars). All infections were performed in triplicate, and the mean luciferase activity (RLU) is shown. The coefficient of variance was <15% in all cases.

Receptor dependency of HIV–HCV pseudotype infectivity. Huh-7.5 cells were incubated with mAbs specific for CD81 (5A6, •), SR-B1 (clone 25, ▴), and an irrelevant isotype-matched IgG (□) at 5 μg/ml and were infected with HIV pseudotypes bearing VSV G (A), H E1E2 (B), and no envelope (data not shown) at varying concentrations of p24 antigen. Infection with HIV-no envelope gave a maximal mean RLU count of 420 ± 50 at 2 ng per well. The coefficient of variance was <10% in all cases.

Soluble CD81 neutralization of HIV–HCV pseudotype infectivity. (A and B) HIV pseudotypes bearing VSV G (A) and H E1E2 (B) at varying concentrations of p24 antigen were incubated with recombinant soluble forms of CD81, GST-humCD81 (▴), and GST-agmCD81 (•)ata final concentration of 1 μg/ml or medium alone (□) for 1 h at 37°C. Virus–CD81 mixtures were tested for infectivity of Huh-7.5 cells. (C) A single dose of HIV-H E1E2 pseudotype (1 ng per well) was incubated with varying concentrations of GST-humCD81 (▴) and GST-agmCD81 (•)for 1 h at 37°C and tested for infection of Huh-7.5 cells. All infections were performed in triplicate, and the mean luciferase activity (RLU) is shown. The coefficient of variance was <10% in all cases.