Hepatitis B Virus genomes associate with cellular sites of DNA damage by inducing replication stress

Hepatitis B Virus (HBV) is a leading cause of liver cancer, with almost 300 million infected individuals worldwide. Although HBV-infected patients benefit from drug regimens that help to control chronic infection, they are rarely clinically cured of HBV. The HBV genome persists in the nucleus of infected hepatocytes in the form of a covalently closed circular DNA (cccDNA) molecule, a reservoir of HBV DNA molecules that serve as the template for reactivation of long-term chronic HBV. However, despite playing a central role in the viral life cycle, little is understood about where cccDNA molecules localize, why they are so stable, and how they impact the host nuclear compartment. Perhaps because of this, there are few treatments that target cccDNA, which is critical for eradication of clinical HBV. Here, we show that HBV infection induces a cellular DNA Damage Response (DDR) that is comparable with cells undergoing replication stress, and this cellular replication stress is initiated after the formation of cellular cccDNA molecules. Using a novel high-throughput chromosome conformation capture technology that monitors the localization of HBV cccDNA molecules, we show that cccDNA molecules persist in the vicinity of many cellular fragile sites. Induction of cellular DNA damage leads to relocalization of the viral HBx oncoprotein to DDR sites in an ATM, ATR and DNA-PK dependent manner. Our findings contribute to the understanding of how HBV cccDNA navigates the host nuclear environment, identifying functional targets for development of therapies against HBV infection and resulting liver cancer.