Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Abstract

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.

Author Summary

Persistent infection with hepatitis B virus (HBV) causes chronic hepatitis B which frequently progresses to hepatocellular carcinoma, a leading cause of cancer-mediated mortality worldwide. Persistence requires formation and amplification of covalently closed circular (ccc)DNA, an episomal form of the viral genome that is not targeted by current drugs and thus is responsible for the notorious difficulties in therapeutic elimination of infection. Initial generation of cccDNA occurs upon nuclear import of the virion-borne relaxed circular (rc) DNA to which the viral polymerase is covalently linked; amplification occurs via intracellular recycling. The underlying molecular pathway is poorly understood. Because HBV infects only primates, in vivo studies are extremely restricted; in vitro, select hepatoma cell lines transfected with HBV support viral replication, however with little if any cccDNA formation. Here, we compared intracellular recycling of HBV and DHBV, a model hepatitis B virus from ducks, in cross-species transfections. Surprisingly, the major contribution to cccDNA formation comes from the virus rather than the cell as DHBV but not HBV rcDNA converted efficiently into cccDNA in the same human cell background. This unexpected difference might help to better understand persistence of HBV infection; efficient DHBV cccDNA formation in human cells provides a new tool to facilitate identification, and possibly targeting, of the human cell factors involved.