The relationship between the cell cycle and early amplification of duck hepatitis B virus covalently closed circular (CCC)
DNA was studied after in vitro
infection of fetal hepatocytes. We first showed that embryonic hepatocytes 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 growth factor alpha (
TGF-alpha). CCC
DNA synthesis increased with progression 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 labeling, viral nucleocapsids were found predominantly in
bromodeoxyuridine-positive hepatocytes, indicating that high viral replication occurs preferentially in proliferating hepatocytes. CCC
DNA was also detected mainly in cells in the S and G2/M phases separated from cells in the G1 phase by cell sorting. Taken together, these results show that hepatocyte proliferation may positively regulate the initial amplification of CCC
DNA of avian hepadnaviruses, and may explain why mitosis is not necessarily associated with loss of CCC
DNA.