FIG. 7.

Nuclear transportation of HBV DP rcDNA and cccDNA formation was inhibited by expression of dominant negative karyopherin-β1 or knockdown of endogenous karyopherin-β1 expression. HepDES19 cells in a collagen-coated 35-mm dish were transfected by Lipofectamine 2000 (Invitrogen) with 4 μg plasmid of control vector (lanes 1 and 4), dominant negative karyopherin-β1 (D.N-kβ1; lane 2), dominant negative karyopherin-α1 (D.N-kα1; lane 3), 90 nM of control siRNA (lane 5; Santa Cruz), or Smartpool siRNA for karyopherin-β1 (lane 6; Dharmacon). The transfected cells were cultured in tetracycline-free medium for 5 days, followed by a second round of transfection and continued culture under tetracycline-free conditions for another 5 days. The intracellular core DNA (A), whole-cell Hirt DNA (B), cytoplasmic DP DNA (C), and nuclear DP DNA (D) were extracted as previously described and subjected to Southern blot and DNA hybridization. The positions of rcDNA (RC), single-stranded DNA (SS), and cccDNA are indicated. Expression of wild-type or recombinant proteins and HBcAg was assessed by a Western blot assay (E).