HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways
HepAD38 cells were treated with 2 mM of PFA from day 2 to day 6 post removal of tet from medium. Intracellular capsids were purified by sucrose gradient centrifugation. (A) Endogenous DNA polymerase reactions with the purified HBV capsids were performed in the absence or presence of dNTP, 2.5 μM of Bay 41–4109, 5 μM of ENAN-34017 or 25 μM of AT-61 at 37°C for 16 h. Three-fourths of each reaction were subjected for extraction of viral DNA without or with prior DNase I digestion at 37°C for 30 min. The viral DNA were detected by Southern blot hybridization with full-length riboprobe recognizing minus strand DNA. The remaining one-fourth of each reaction was analyzed by a particle gel assay to detect HBV capsids. (B) Endogenous DNA polymerase reactions were performed in the presence of dNTP and indicated concentrations of Bay 41–4109, ENAN-34017 or AT-61 at 37°C for 16 h. Viral DNA and capsids were analyzed as described above. The relaxed circular (RC) DNA, double-stranded linear (DSL) DNA and full-length single stranded (SS) DNA species were indicated.