FIG. 5.

Purification of HCV-like particles by sucrose and CsCl gradient centrifugation. (A) Sucrose and CsCl equilibrium centrifugation. At 96 h postinfection, insect cells infected with BVHCV.S were lysed and subjected to low-speed centrifugation, and the supernatant was pelleted over a 30% sucrose cushion. The pellet containing the viruslike particles was resuspended and subjected to a second sucrose or CsCl equilibrium gradient centrifugation as described in Materials and Methods. Ten fractions were collected from the top and analyzed by SDS-PAGE and immunoblotting with anti-core (C), anti-E1, or anti-E2 antibodies. Molecular masses (in kilodaltons) of protein molecular weight (MW) markers are indicated on the left; HCV-specific proteins are indicated on the right. (B) Sucrose sedimentation velocity centrifugation. Lysates of insect cells infected with BVHCV.S and BVHCV.Sp7− were layered onto a 10 to 60% sucrose gradient and centrifuged for 2.5 h at 4°C and 200,000 × g. Ten fractions were collected from the top and analyzed for HCV structural proteins as described above.