Fig. 4.

HBc expression is not required for the maintenance of cccDNA levels over months of infection.

(A) HepG2-NTCP cells were infected with WT or ΔHBc HBV at mge 40. Cells were fixed 1, 4, and 9 weeks post inoculation and immunofluorescence for HBs and HBc was performed. Scale bar represents 100 μm. Cells infected with WT or ΔHBc HBV were harvested every week for total DNA extraction. (B) cinqPCR was then performed to quantify cccDNA (left) and total HBV DNA (right) levels relative to the single-copy cellular gene RNaseP. MyrB treatment was used as control for infection inhibition (1, 4, and 9 weeks post inoculation). Two biological replicates (thin lines) were carried out. Error bars (Poisson 95% CI) represent the technical error of the ddPCR assay. The mean value of the 2 replicates is shown as a thick line. Results are representative of 2 independent experiments. cccDNA, covalently closed circular DNA; HBV, hepatitis B virus; mge, multiplicity of genomic equivalents; NTCP, sodium taurocholate cotransporting polypeptide; WT, wild-type.