Distinct dual antiviral mechanism that enhances hepatitis B virus mutagenesis and reduces viral DNA synthesis

Reverse transcriptase (RT) is an essential enzyme for the replication of retroviruses and hepadnaviruses. Current therapies do not eliminate the intracellular viral replication intermediate termed covalently closed circular (ccc) DNA, which has enhanced interest in hepatitis B virus (HBV) reverse transcription and cccDNA formation. The HBV cccDNA is generated as a plasmid-like episome in the host cell nucleus from the protein-linked relaxed circular (rc) DNA genome in incoming virions during HBV replication. The creation of the cccDNA via conversion from rcDNA remains not fully understood. Here, we sought to investigate whether viral mutagens can effect HBV replication. In particular, we investigated whether nucleoside analogs that act as viral mutagens with retroviruses could impact hepadnaviral DNA synthesis. We observed that a viral mutagen (e.g., 5-aza-2′-deoxycytidine, 5-aza-dC or 5-azacytidine, 5-aza-C) severely diminished the ability of a HBV vector to express a reporter gene following virus transfer and infection of target cells. As predicted, the treatment of 5-aza-dC or 5-aza-C elevated the HBV rcDNA mutation frequency, primarily by increasing the frequency of G-to-C transversion mutations. A reduction in rcDNA synthesis was also observed. Intriguingly, the cccDNA nick/gap region transcription was diminished by 5-aza-dC, but did not enhance viral mutagenesis. Taken together, our results demonstrate that viral mutagens can impact HBV reverse transcription, and propose a model in which viral mutagens can induce mutagenesis during rcDNA formation and diminish viral DNA synthesis during both rcDNA formation and the conversion of rcDNA to cccDNA.