Figure 4. Replication efficiency of BsdR and hrGFP HBV vectors.

HepG2 cells (A) or Huh7 cells (B) were transfected with plasmid pCH-9/3093 (1), pCH-BsdR (2) or pCH-hrGFP (3). Extracellular and cytoplasmic HBV DNA was extracted 4 days post-transfection. Replicative intermediates were monitored by Southern blotting using α ³²P-labeled HBV probe, or a mixture of BsdR- and hrGFP-specific probes (A, lower panel). The positions of RC- and dsL HBV DNA are indicated. To reveal the weak signals from the pCH-hrGFP construct, long exposures of the same blots are also shown. For the BsdR construct, the signals for total DNA, and separately for RC-DNA were quantitated by phosphorimaging and related to the signal intensities from the wild-type HBV samples (set at 100%); mean values from three experiments are indicated at the bottom of the blots. Both the total amount of replicative DNA and RC-DNA signal intensity were significantly different between wild-type constructs and BsdR in both HepG2 and Huh7 cells (P<0.01). The transgene-specific probes (A, lower panels) detected additional faster migrating species in the transgene-related samples, including distinct bands whose mobility slightly differed between BsdR and hrGFP vector (labeled with asterisks); these might represent reverse transcription products from small amounts of spliced recombinant pgRNAs (see text for details). In addition, bulk viral DNA associated with capsids was visualized by hybridization with a ³²P-labeled HBV DNA probe following separation of the capsids by NAGE (small panels). The qPCR results for extracellular HBV DNA are shown in the bar graphs on the right. Copy numbers are given as log10 per 1 ml of culture supernatant, collected from 72 h–96 h post transfection. The mean values from three independent experiments including SD are shown. All differences were significant, with P<0.05 for BsdR vector vs. wild-type HBV vector, and P<0.01 for the others.