FIG. 7.

Characterization of HDV RNA-containing particles by isopycnic centrifugation in a cesium chloride gradient. Culture medium from HuH-7 cells was collected at days 6, 9, 12, and 15 after transfection of 0.5 × 10⁶ cells with 0.6 μg of pSVLD3 plasmid DNA and 1.4 μg of WT, env-negative (EN), or S protein mutant DNA. Particles were concentrated from 5 ml of culture medium and subjected to centrifugation to equilibrium in 12-ml cesium chloride gradients. Fractions were collected from the bottom of the tube, and proteins from 1/4 of each 1-ml fraction were analyzed by immunoblotting with anti-S antibodies (1:2,000). RNA was extracted from each fraction for analysis by agarose gel electrophoresis and blot hybridization by using a genomic strand-specific ³²P-labeled HDV RNA probe. The gel migration positions of genomic HDV RNA (1.7 kb) and S proteins (p24 and gp27) are indicated. The numbering (4 to 11) of each fraction is indicated. Fractions 8 and 9 correspond to fractions with densities of 1.22 and 1.18 g/cm³, respectively. Purified WT HDV particles were used as controls (C).