Despite the utmost importance of cccDNA in HBV biology, the mechanism by which cccDNA synthesis is regulated is not completely understood. Here we explored HepG2-NTCP cell line and performed a time-course HBV infection experiment (up to 30 days) to follow the conversion of the input viral DNA into cccDNA. We found that a protein-free RC DNA (PF-RC DNA) become detectable as early as 12 h post infection (hpi) prior to the detection of cccDNA, which become evident only at 2–3 dpi. Intriguingly, the PF-RC DNA detected at 12 hpi was abundantly located in the cytoplasm, implicating that the protein-removal from the input viral DNA takes place in the cytoplasm, perhaps inside the nucleocapsid. Notably, during the early time points of HBV infection, the PF-RC DNA accumulated at significantly higher levels and appeared in a peak followed by a plateau at late time points with dramatically lower levels, implicating the presence of two distinct populations of the PF-RC DNA. Importantly, the PF-RC DNA at earlier peak is entecavir (ETV)-resistant, whereas the PF-RC DNA at posterior days is ETV-sensitive. An interpretation is that the PF-RC DNA at earlier peak represents “input viral DNA” derived from HBV inoculum, whereas the PF-RC DNA at late time points represents the de novo product of the viral reverse transcription. The existence of two populations of the PF-RC DNA having a distinct kinetic profile and ETV-sensitivity implicated that intracellular amplification via the viral reverse transcription greatly contributes to the maintenance of cccDNA pool during HBV infection. As such, we concluded that the cccDNA level is stably maintained by continuing replenishment of cccDNA primarily through intracellular amplification in the HepG2-NTCP cell line.
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