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

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          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.

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          Most cited references53

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          Control of cccDNA function in hepatitis B virus infection.

          The template of hepatitis B virus (HBV) transcription, the covalently closed circular DNA (cccDNA), plays a key role in the life cycle of the virus and permits the persistence of infection. Novel molecular techniques have opened new possibilities to investigate the organization and the activity of the cccDNA minichromosome in vivo, and recent advances have started to shed light on the complexity of the mechanisms controlling cccDNA function. Nuclear cccDNA accumulates in hepatocyte nuclei as a stable minichromosome organized by histone and non-histone viral and cellular proteins. Identification of the molecular mechanisms regulating cccDNA stability and its transcriptional activity at the RNA, DNA and epigenetic levels in the course of chronic hepatitis B (CH-B) infection may reveal new potential therapeutic targets for anti-HBV drugs and hence assist in the design of strategies aimed at silencing and eventually depleting the cccDNA reservoir.
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            Global control of hepatitis B virus infection.

            Worldwide about 350 million people are chronic carriers of the hepatitis B virus (HBV). The infection can cause acute and chronic liver disease including cirrhosis and hepatocellular carcinoma (HCC). Hepatocellular injuries of HBV infection are predominantly immune-mediated, and the natural history of chronic infection can be divided into three phases based on virus-host interactions-namely, immune tolerance, immune clearance, and viral integration phases. Four serotypes (adw, ayw, adr, and ayr) and seven genotypes (A to G) of HBV have been identified, and they show some distinct geographic distributions. The HBV genotypes may have clinical relevance and are currently under investigation. On the basis of disease burden and the availability of safe and effective vaccines, the WHO recommended that by the end of the 20th century hepatitis B vaccine be incorporated into routine infant and childhood immunisation programmes for all countries. The efficacy of universal immunisation has been shown in different countries, with striking reductions of the prevalence of HBV carriage in children. Most important, hepatitis B vaccination can protect children against HCC and fulminant hepatitis, as has been shown in Taiwan. Nevertheless, the implementation of worldwide vaccination against HBV requires greater effort to overcome the social and economic hurdles. Safe and effective antiviral treatments are available but are still far from ideal, a situation that, hopefully, will be improved soon. With hepatitis B immunisation, the global control of HBV infection is possible by the end of the first half of 21st century.
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              Hepatitis B virus replication.

              Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, epsilon, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV), now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cell-free systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the epsilon RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                September 2010
                September 2010
                2 September 2010
                : 6
                : 9
                Affiliations
                [1]Department of Medicine II, University Hospital of Freiburg, Freiburg, Germany
                University of Southern California, United States of America
                Author notes

                Conceived and designed the experiments: JK MN CT. Performed the experiments: JK CR JJZ. Analyzed the data: JK HEB MN CT. Wrote the paper: JK MN CT.

                Article
                10-PLPA-RA-3777R1
                10.1371/journal.ppat.1001082
                2932716
                20824087
                5dd4e434-af6a-4414-9ec6-15727a53ea25
                Köck et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 15
                Categories
                Research Article
                Gastroenterology and Hepatology/Hepatology
                Infectious Diseases/Viral Infections
                Molecular Biology

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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