Initial amplification of duck hepatitis B virus covalently closed circularDNA after in vitro infection of embryonic duck hepatocytes is increased bycell cycle progression

The relationship between the cell cycle and early amplification of duck hep atitis B virus covalently closed circular (CCC) DNA was studied after in vi tro infection of fetal hepatocytes. We first showed that embryonic hepatocy tes proliferated for at least 6 days after plating and that complete viral replication including CCC DNA amplification occurred in these proliferating cells. Addition of sodium butyrate or aphidicolin reversibly blocked cells in the G1 phase and diminished CCC DNA synthesis, which was restored after drug withdrawal, concomitantly with the entry of cells into S phase. Cell cycle progression of fetal hepatocytes can be triggered by stimulation with epidermal growth factor (EGF), hepatocyte growth factor (HGF), and tumor g rowth factor alpha (TGF-alpha). CCC DNA synthesis increased with progressio n to the S phase induced by EGF, HGF, and TGF-alpha alone or in combination . By contrast, tumor growth factor beta (TGF-beta) alone or in combination with EGF inhibited cell proliferation and viral DNA synthesis. By double la beling, viral nucleocapsids were found predominantly in bromodeoxyuridine-p ositive hepatocytes, indicating that high viral replication occurs preferen tially in proliferating hepatocytes. CCC DNA was also detected mainly in ce lls in the S and G2/M phases separated from cells in the G1 phase by cell s orting. Taken together, these results show that hepatocyte proliferation ma y positively regulate the initial amplification of CCC DNA of avian hepadna viruses, and may explain why mitosis is not necessarily associated with los s of CCC DNA.