HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways
C3AhNTCP cells were infected with HBV at an MOI of 500 genome equivalents. The cells were mock-treated or treated with 100nM of MyrB, 1 μM of ETV, 2.5 μM of Bay 41–4109, 1 μM of GLS-4, 5 μM of ENAN-34017 or 1,000 IU/ml of IFN-α, starting from 24 h before infection until harvesting at 3 or 6 days post infection (dpi). HBV cccDNA (A), pgRNA (B) and cytoplasmic core DNA (C) were quantified by real-time PCR assays. Differences in viral cccDNA, core DNA or pgRNA between mock-treated control and treated groups were statistically analyzed (t-test, * p <0.05, ** p <0.01, *** p <0.001). (D) Hybridization analyses of HBV replication intermediates in cells harvested at 6 dpi. Upper panel, Hirt DNA extracted from the cells harvested at 6 dpi were denatured at 88°C for 5 min to denature DP-rcDNA to single-stranded DNA and followed by restriction with EcoRI to convert cccDNA into unit-length double stranded linear DNA and detected by Southern blot hybridization (labeled as CCC/EcoRI). Unit-length HBV linear DNA served as a molecular weight marker. Lower panel, HBV RNAs, pre-genomic RNA (pgRNA), 2.4 and 2.1kb mRNA specifying envelope proteins were determined by Northern blot hybridization. 28S and 18S ribosomal RNA (rRNA) served as loading controls.