To generate HCV pseudo-particles, 293T cells were transfected with expression vectors encoding the viral components (see Fig. 1 B), i.e., E1E2 glycoproteins, retroviral core proteins, and packaging-competent GFP- or nlslacZ-containing retroviral transfer vectors. In brief, the Gag-Pol packaging construct (8.1 µg), the transfer vector construct (8.1 µg), and the glycoprotein-expressing construct (2.7 µg) DNAs were transfected into 2.5 × 106 293T cells seeded the day before in 10-cm plates using a calcium phosphate transfection protocol (CLONTECH Laboratories, Inc.), as described previously (11 (link)). The medium (8 ml/plate) was replaced 16 h after transfection. Supernatants containing the pseudo-particles were harvested 24 h later, filtered through 0.45-µm pore-sized membranes, and used in infection assays. Purified virus samples were obtained by ultracentrifugation of 10-ml viral supernatants through a 1.5-ml 20% sucrose cushion in an SW 41 Beckman rotor (25,000 rpm, 2.5 h, 4°C). Viral pellets were suspended in 50 µl PBS. Immunoblots of producer cell lysates and purified pseudo-particles were performed as described previously (15 (link)). Fractionation of the sucrose cushion purified viral pellets was achieved by an overnight equilibrium density centrifugation in a 20–60% sucrose gradient at 35,000 rpm and 4°C in a Beckman SW 41 rotor. Fractions of 0.7 ml were collected, precipitated with TCA, and analyzed by Western blotting.