FIG 2.

Analysis of HBV DNA synthesis in HBV-Met.4 cells. (A) HBV-Met.4 cells were differentiated with 2% DMSO up to 15 days to induce HBV gene expression and replication. The viral core DNA (lanes 1 to 3) and PF DNA (lanes 4 to 6) were isolated every 5 days and detected by Southern blotting. The PF DNA from day 15 postinduction was further heat treated (100°C) to denature non-CCC DNA species, which was followed by EcoRI digestion to linearize CCC DNA (lane 8). Lane 7 was a much longer exposure than lane 6 and had the same exposure time as lane 8. (B) HBV-Met.4 cells, without DMSO induction, were transiently transfected with pCMV-HBV/WT (lanes 2, 4, and 6) or pCMV-HBV/Pol⁻ (lanes 1, 3, and 5). Five days later, the viral core DNA (lanes 1 and 2) and PF DNA (lanes 3 and 4) were isolated and detected by Southern blotting. DpnI was used to digest transfected plasmid DNA that was coisolated with HBV PF DNA prior to Southern blot analysis of all PF DNA. The PF DNA was further heat denatured (100°C), followed by EcoRI digestion to linearize the CCC DNA (lanes 5 and 6). Lanes 7 and 8 represent a longer exposure of lanes 5 and 6. RC, relaxed circular; CCC, covalently closed circular; SS, single stranded; DpnI fragments, plasmid DNA fragments generated by DpnI digestion.