Identifying the role of DNA ligase I in hepatitis B virus cccDNA formation in human hepatoma cells

Abstract

Over 257 million people are chronically infected with hepatitis B virus (HBV) worldwide, and these patients are at a 100-fold higher risk for developing cirrhosis and hepatocellular carcinoma compared to the general population. Current treatment can only suppress the virus but rarely lead to a cure. Limitations to current treatment options are largely due to the persistence of the viral covalently closed circular DNA (cccDNA)—the critical viral replication intermediate. While previous studies have identified cccDNA elimination as the key to curing HBV infection, the mechanism for cccDNA formation and maintenance remains to be fully elucidated. Recent findings identified a minimal set of host factors necessary for cccDNA formation by biochemically reconstituting this reaction. To validate these findings in a cellular environment that more closely resembles the native biological context of an HBV infection, we first established a system to rapidly and conditionally deplete DNA ligase I, one of the five identified host factors, in HBV-susceptible human hepatoma cells by dually introducing CRISPR knockout and the degron system. Following HBV infection in this tissue culture model, we observed that neither LIG1 overexpression nor partial knockout demonstrated any effects on cccDNA levels, and the challenges of establishing a complete knockout precluded us from drawing definitive conclusions on the role LIG1 plays in cccDNA formation. The findings in this study extend the applicability of the degron system to studying the role of essential host factors in viral-host interactions and contribute to current work in decoding the mechanisms of cccDNA formation. By establishing an experimental framework to interrogate the effects of essential host factors in cccDNA formation, this study lays the foundation for identifying promising therapeutic targets for chronic HBV patients.