Overexpression of Tdp2 was associated with a decrease in HBV CCC DNA.

<p>(A) and (B). HEK293 cells in 6 cm dishes were cotransfected with the envelope-defective HBV (5 μg) and Tdp2 (+) or empty vector (-) (5 μg) plasmid. Five days post-transfection the cells were harvested and analyzed for protein expression and viral DNA levels. (A) HBV core DNA was extracted and analyzed by Southern blotting (top panel). Tdp2 and HBc levels were measure by western blotting (2nd and 3rd panels). β<b>-</b>actin was measured by western blotting as a loaded control (4^th panel). (B) HBV PF DNA were extracted and analyzed by Southern blotting. PF DNA samples were Dpn I-digested to degrade plasmid DNA (lanes 1–4). Plasmid fragments at the bottom of the agarose gel are indicated. To further verify the identity of CCC DNA, the Dpn I digests were boiled to reduce RC and double-stranded linear (DSL) DNA species (as well as plasmid fragments) to single strands whereas the supercoiled CCC DNA remained circular (lanes 5–8), or boiled and then digested to linearize the CCC DNA (lanes 9–12). PF DNA extracted from HepAD38 cells was loaded in lane 13 as a positive control for the mobility of the RC, DSL, and CCC DNA species. (C) Quantitative analysis of HBV core and PF DNA. The triangles at the bottom represent the graded amounts of HBV and Tdp2 (+) or empty vector (-) plasmid: left bar, 7.5 μg HBV plasmid and 2.5 μg Tdp2 (+) or empty vector (-) plasmid; middle bar, 5 μg each HBV and Tdp2 (+) or empty vector (-) plasmid; right bar, 2.5 μg HBV plasmid and 7.5 μg Tdp2 (+) or empty vector (-) plasmid. The indicated viral DNA ratios were normalized to the corresponding DNA ratios from empty vector transfection (set to 100%) and are reported as the mean relative ratios ± standard error of the mean of three independent experiments. The asterisks (*) indicate a significant difference in CCC/core RC at HBV:Tdp2 plasmid transfection ratios of 5 μg: 5μg and 2.5 μg: 7.5 μg, compared to corresponding HBV:empty vector transfections (P < 0.05; unpaired, two-tailed t-test). CCC, CCC DNA; DSL, DSL DNA; RC, RC DNA.