HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways
As depicted in panel (A), HepAD38 cells were treated with 2 mM of PFA from day 2 to day 6 after removal of tet from medium. On day 6, PFA-containing medium were removed and the cells were mock-treated or treated with 1 μM of ETV, 2.5 μM of Bay 41–4109 or 5 μM of ENAN-34017 and harvested at 0, 3, 6, 9 and 12 h of the treatment. The cytoplasmic core DNA (B), cccDNA and DP-rcDNA (C) were analyzed by Southern blot hybridization. The relaxed circular (RC) DNA, double-stranded linear (DSL) DNA and full-length single stranded (SS) DNA species were indicated. For cccDNA and DP-rcDNA analysis, before loading, Hirt DNA were denatured at 88°C for 5 min, which denatures DP-rcDNA into single-stranded DNA (labeled as denatured DP-rc).