FIG. 9.
HBV cytoplasmic DP DNA cosediments with nucleocapsids. Three milliliters of cytoplasmic fraction prepared from 2 × 107 HepDES19 cells cultured in the absence of tetracycline for 10 days was overlaid on 33 ml of a 10 to 60% (wt/vol) sucrose gradient and centrifuged at 24,000 rpm for 16 h at 4°C using a Beckman SW28 rotor. Seventeen 2.25-ml fractions were collected from the bottom. HBV core antigen (A), total HBV DNA (B), and capsid DNA (C) in each fraction were assayed as described in Material and Methods. DP-rcDNA was extracted from each fraction without (D) or with (E) prior DNase I digestion. For the immunoprecipitation (IP) assay, each faction was mixed with Sepharose 4B-protein A beads preabsorbed with rabbit anti-HBc and incubated at 4°C overnight. Beads were washed three times with TNE buffer, and protein-free DNA was extracted without (F) or with (G) prior DNase I digestion. The Hirt DNAs were resolved in a 1.2% agarose gel and detected by Southern blot hybridization. RC, rcDNA; SS, single-strand DNA; EIA, enzyme immunoassay.