Abstract
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 α) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol α by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol α expression by siRNA led to suppression of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol α genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol α to disrupt cccDNA biosynthesis. Mechanistically, Pol α is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol α 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 κ and Pol λ.
Highlights
Hepatitis B virus (HBV) chronically infects 257 million people worldwide [1]
Understanding the biosynthesis and maintenance of cccDNA minichromosome is crucial for the development of novel antiviral therapeutics to cure chronic HBV infection
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
Summary
Hepatitis B virus (HBV) chronically infects 257 million people worldwide [1]. Chronic HBV carriers have a higher risk of developing cirrhosis and hepatocellular carcinoma (HCC), which accounts for approximately 686,000 annual deaths [1]. Given the unique structure of rcDNA, the conversion of rcDNA to cccDNA requires at least four steps: (i) the completion of plus strand DNA synthesis by DNA polymerases; (ii) removal of the viral polymerase covalently attached at the 5’ end of minus strand DNA and the capped RNA primer at the 5’ end of plus strand DNA via unknown mechanisms; (iii) processing of the ends of both strands of rcDNA by cellular nucleases; and (iv) filling in the gaps by host DNA polymerases and ligation of DNA ends by ligases [15]. Our recent work showed that treatment with HBV core protein allosteric modulators (CpAMs) to induce premature uncoating of viral nucleocapsids led to reduced cccDNA formation in de novo infection, but increased cccDNA synthesis
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