FIG. 2.
HCV nucleocapsid-like particle consists of core complex. (a) HCV RNA titer in culture medium separated on a 20 to 50% sucrose density gradient. Concentrated culture medium from JFH1E2FL RNA-transfected HuH-7 cells were treated with RNase and separated on a 20 to 50% sucrose density gradient. Fractions 1 to 16 were obtained from the bottom to the top of the tube, respectively. The HCV RNA titer and infectivity of each fraction were analyzed by real-time qRT-PCR (for fractions 1 to 16) and counting the number of cells infected with HCV-like particle detected by immunofluorescence (for fractions 3 to 14), respectively, as described in Materials and Methods. In brief, each fraction was diluted with 1× PBS and HCV-like particles were collected by ultracentrifugation, and then the pellets were suspended in culture medium and used for infection. (b) HCV-like particle collected from the infectious HCV peak (from panel a, filled arrowhead) and the HCV RNA peak (from panel a, open arrowhead) were collected by ultracentrifugation, subjected to nonreducing SDS-PAGE, and detected by immunoblotting against the core protein. (c) HCV-like particles collected from fractions 8 to 13 (a) were subjected to nonreducing SDS-PAGE in the presence (lane +) or absence (lane −) of 5 mM NEM and analyzed by immunoblotting against the core protein. Data are representative of two (a, infectivity of fractions) or three independent experiments.