Replication-driven HBV cccDNA loss in chimeric mice with humanized livers.

Hepatitis B virus (HBV) infection depends on the establishment of covalently closed circular DNA (cccDNA) and can be either transient or persistent. Persistent infection requires a noncytopathic viral phenotype, primarily achieved by limiting replication in infected cells. This study aimed to understand how most HBV-infected cells can avoid cytopathic consequences despite robust replication. Using chimeric mice with humanized livers, we assessed HBV replication kinetics and observed a progressive accumulation of viral products from early stages up to peak infection in infected cells, but the accumulation stopped increasing upon reaching the persistence phase, possibly resulting from the inhibition of HBV replication. We then examined HBV products, including cccDNA, transcription, and viral protein levels, to identify the potential point of inhibition, finding no clear suppression of viral transcription or protein synthesis. Quantitative analysis of cccDNA levels in bulk cells, single nuclei, and individual HBsAg-positive cells revealed that cccDNA was undetectable in a portion of infected cells. Our findings raise the possibility that infected cells may spontaneously clear cccDNA. This would stop HBV replication at its root and avoid the potential cytopathic consequences in infected cells due to uncontrolled viral replication. These results suggest that efficient cccDNA elimination might be achievable without directly targeting existing cccDNA.IMPORTANCEThe primary barrier to curing chronic HBV infection is the persistence of covalently closed circular DNA (cccDNA), which is traditionally considered stable within infected cells. However, clinical observations have revealed that cccDNA can undergo frequent clearance and replacement in patients with chronic HBV infection. Building on these observations, our study demonstrated that cccDNA was undetectable in a portion of HBV-infected cells at different timepoints after peak infection in chimeric mice with humanized livers, suggesting that spontaneous cccDNA clearance may occur. These findings align with clinical data and indicate that effective cccDNA elimination may be possible without the need to target cccDNA itself directly.