DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA

Persistent hepatitis B virus (HBV) infection relies on the establishment and maintenance of covalently closed circular (ccc) DNA, a 3.2 kb episome that serves as a viral transcription template, in the nucleus of an infected hepatocyte. Although evidence suggests that cccDNA is the repair product of nucleocapsid associated relaxed circular (rc) DNA, the cellular DNA polymerases involving in repairing the discontinuity in both strands of rcDNA as well as the underlying mechanism remain to be fully understood. Taking a chemical genetics approach, we found that DNA polymerase alpha (Pol ) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol expression by siRNA led to suppression of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol to disrupt cccDNA biosynthesis. Mechanistically, Pol is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol is involved in the repair of the minus strand DNA nick in cccDNA synthesis. Our study thus reveals that the distinct host DNA polymerases are hijacked by HBV to support the biosynthesis of cccDNA from intracellular amplification pathway compared to that from de novo viral infection, which requires Pol and Pol . Author summary CCC DNA is the most refractory HBV replication intermediate under long-term antiviral therapies and is responsible for the viral rebound after treatment cessation. Therefore, understanding the biosynthesis and maintenance of cccDNA minichromosome is crucial for the development of novel antiviral therapeutics to cure chronic HBV infection. Although it has been clearly demonstrated that cccDNA biosynthesis relies on host cellular DNA repair machinery, the molecular pathways that convert rcDNA into cccDNA remain to be identified. Here we report that DNA polymerase alpha (Pol ) as well as Pol and are required for converting rcDNA into cccDNA through intracellular cccDNA amplification. This finding adds novel molecular insights on cccDNA biosynthesis. Further understanding the mechanism of cccDNA synthesis should reveal molecular targets for developing therapeutic agents to eradicate cccDNA and cure chronic hepatitis B.