Chronic hepatitis B virus (HBV) infection is typically lifelong due to the persistence of the viral episomal covalently closed circular (ccc)DNA, from which all viral products can be produced. In vitro studies show that cccDNA is lost after cell mitosis, resulting in uninfected daughter cells. However, even with extensive liver turnover and viral suppression, HBV persists in animal models and patients. This study investigates how cccDNA is maintained in in vitro and in vivo models of HBV.
HBV-infected HepG2-NTCP cells were passaged to induce mitosis. We found that cccDNA levels decreased at rates consistent with loss with mitosis for ~3 doublings, but then plateaued at ~1:500 of initial values. The cccDNA-positive cells after extensive passaging expressed p21 and beta-galactosidase, indicating mitotic arrest. Using a reporter HBV expressing red fluorescent protein, we isolated these cells with FACS. Characterisation of these cells with RNA sequencing revealed upregulation of pathways associated with cell cycle arrest. A panel of drugs targeting mitotic arrest pathways was screened and candidates identified which effectively induced up to a 12-fold reduction in cccDNA levels.
We tested these drugs in our in vivo model of episomal DNA persistence: C3H mice were transduced with a green fluorescent protein (GFP)-expressing adeno-associated virus vector (a surrogate for HBV cccDNA), and liver turnover was induced with twice-weekly carbon tetrachloride injections for three weeks. Immunofluorescence revealed a subset of hepatocytes which retained GFP and were found to express p53 and p21. Treatment of these mice with drug candidates resulted in a ~3-fold reduction in these GFP retaining cells.
Thus, we identify a novel population of cells that potentially retain cccDNA through mitotic arrest, which may explain the persistence of cccDNA. Targeting these reservoir cells may be crucial for developing novel cure therapies for chronic hepatitis B.