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.

The carboxy-terminal sequence of the hepatitis B virus (HBV) core protein constitutes a nucleic acid binding domain that is rich in arginine residues and contains three serine phosphorylation sites. While dispensable for capsid assembly, this domain is involved in viral replication, as demonstrated by the effects of mutations on RNA packaging and/or reverse transcription; however, the underlying mechanisms are poorly understood. Here we tested a series of core protein mutants in which the three serine phosphorylation sites were replaced by glutamic acid, in parallel with a previously described deletion variant lacking the 19 C-terminal amino acid residues, for their ability to support viral replication in transfected hepatoma cells. Replacement of all serines and the deletion gave rise to nucleocapsids containing a smaller than wild-type DNA genome. Rather than a single-stranded DNA intermediate, as previously thought, this was a 2.0-kbp double-stranded DNA molecule derived from spliced pregenomic RNA (pgRNA). Interestingly, full-length pgRNA was associated with nucleocapsids but was found to be sensitive to nuclease digestion, while encapsidated spliced RNA and 3 truncated RNA species were nuclease resistant. These findings suggest that HBV pgRNA encapsidation is directional and that a packaging limit is determined by the C-terminal portion of the core protein.Hepatitis B virus (HBV) is an important human pathogen accounting for about one million deaths each year (13). The viral genome, as present in infectious virions, is a 3.2-kbp circular, partially double-stranded DNA (dsDNA) molecule that contains overlapping reading frames encoding the core protein, a reverse transcriptase (P), three surface proteins, and the X protein (20,25).Viral replication involves reverse transcription of a pregenomic RNA (pgRNA) intermediate inside nucleocapsids, which are formed by 180 or 240 core protein subunits (4-6, 30). Specific encapsidation of pgRNA occurs via binding of P protein to a 5Ј-proximal RNA stem-loop structure, termed ε. Subgenomic RNAs lack the 5Ј ε and are excluded from encapsidation. Notably, however, spliced RNA species containing all sequence elements necessary for packaging and for reverse transcription have been observed in liver tissue and in transfected cells (9,23,(26)(27)(28)(29). Viral DNA synthesis is a highly complex process involving three translocation events of P to priming sites located at the respective 5Ј and 3Ј ends of the template (17). Thus, both ends must be accessible to the encapsidated P protein. The final product is a partially doublestranded, relaxed circular DNA (rcDNA). Occasionally, one of the translocation steps fails (in situ priming), resulting in a double-stranded linear molecule (17,19,24,25).The C-terminal domain of the core protein plays a crucial role in viral replication. It is rich in arginine residues and contains three serine phosphorylation sites. Though dispensable for particle assembly, it is required for pgRNA packaging (2,3,7,10). Further, the phenotypes of core pr...

APOBEC3G is a cellular cytidine deaminase displaying broad antiretroviral activity. Recently, it was shown that APOBEC3G can also suppress hepatitis B virus (HBV) production in human hepatoma cells. In the present study, we characterized the mechanisms of APO-BEC-mediated antiviral activity against HBV and related hepadnaviruses. We show that human APOBEC3G blocks HBV production in mammalian and nonmammalian cells and is active against duck HBV as well. Early steps of viral morphogenesis, including RNA and protein synthesis, binding of pregenomic RNA to core protein, and self-assembly of viral core protein, were unaffected. However, APOBEC3G rendered HBV core protein-associated full-length pregenomic RNA nuclease-sensitive. Ongoing reverse-transcription in capsids that had escaped the block in morphogenesis was not significantly inhibited. The antiviral effect was not modulated by abrogating or enhancing expression of the accessory HBV X protein, suggesting that HBV X protein does not represent a functional homologue to the HIV vif protein. Furthermore, human APOBEC3F but not rat APOBEC1 inhibited HBV DNA production. Viral RNA and low-level DNA produced in the presence of APOBEC3F or rat APOBEC1 occasionally displayed mutations, but the majority of clones were wild-type. In conclusion, APOBEC3G and APOBEC3F but not rat APOBEC1 can downregulate the production of replication-competent hepadnaviral nucleocapsids. In contrast to HIV and other retroviruses, however, APOBEC3G/3F-mediated editing of nucleic acids does not seem to represent an effective innate defense mechanism for hepadnaviruses. H epadnaviruses are a group of small enveloped DNA viruses with a narrow host range and a relative tropism for the liver. Hepatitis B virus (HBV), the prototypic member of the hepadnavirus family, is a major cause of liver disease worldwide, ranging from acute and chronic hepatitis to cirrhosis and hepatocellular carcinoma. 1,2 Other members of the family include the duck hepatitis B virus (DHBV) and the woodchuck hepatitis virus. 3,4 Hepadnaviral replication involves reverse-transcription of a pregenomic RNA (pgRNA) intermediate inside nucleocapsids, which are formed by 180 or 240 core protein subunits. Inside the capsid, the viral polymerase converts pgRNA into minus-strand DNA, which in turn is completed to a double-stranded, relaxed circular DNA molecule. 5,6 This life cycle places HBV into the large family of retroelements, which all require reverse-transcription of an RNA intermediate.Recently, a cellular defense mechanism targeting a wide range of retroviruses was identified. It was shown that the propagation of HIV-1 strains lacking the accessory protein Vif is suppressed in a number of nonpermissive cells and that this block was due to expression of the cytidine deaminase APOBEC3G (A3G). 7,8 Further studies revealed that A3G induces massive C3 U deamination of single stranded retroviral DNA, resulting in DNA degradation or lethal G3 A hypermutation. [9][10][11] Interestingly, A3G can also interfere with the HBV life c...

The Asian tree shrew, Tupaia belangeri, has been proposed as a novel animal model for studying hepatitis B virus (HBV) infection. Here, we describe a protocol for efficient and reproducible infection of primary tupaia hepatocytes with HBV. We report that human serum interferes with HBV binding to the hepatocytes, thus limiting the maximum multiplicity of infection. Purification of HBV virions by gradient sedimentation greatly enhances virus binding and infectivity. Covalently closed circular DNA was clearly detectable by Southern blot analysis and newly synthesized single-stranded HBV DNA was visible 2 weeks postinoculation. Primary tupaia hepatocytes are also susceptible to infection with the recently discovered woolly monkey hepatitis B virus (WMHBV) but not to woodchuck hepatitis virus infection. Compared to HBV, WMHBV replicated at a higher rate with single-stranded DNA detectable within the first week postinoculation. Primary tupaia hepatocytes should represent a useful system for studying early steps of HBV and WMHBV infection.With an estimated 350 million chronically infected people worldwide, hepatitis B virus (HBV) infection represents a major health care problem. Every year nearly 1 million individuals succumb to HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma (9, 18). Although an efficient and safe vaccine is available, HBV is spreading, especially in Asia and Africa (10). Treatment of chronic HBV infection is still unsatisfactory. At present, alpha interferon and lamivudine are the only therapeutic options available in clinical practice. However, alpha interferon yields a sustained suppression of viral replication in only about one-third of patients, and lamivudine resistance with mutations in the viral polymerase gene is a frequent event in lamivudine-treated patients (3, 7). Based on transfection studies of hepatoma cell lines, the mechanisms of HBV replication have been elucidated in great detail (11,13). By contrast, little is known about the early events of the viral life cycle. Unfortunately, permanent cell lines are not permissive to HBV infection, and primary human hepatocytes are not easily available for in vitro infection studies. Furthermore, the quality of liver tissue obtained at surgery for the preparation of primary human hepatocytes is highly variable (5). Therefore, alternative experimental systems for studying HBV infection are urgently needed.Two reports have described transient HBV infection of the Asian tree shrew, Tupaia belangeri, in vivo (17,20). Furthermore, successful infection of tupaias with human herpes simplex virus and hepatitis C virus has been described (2, 19). Tupaias are squirrel-like animals that are closely related to primates and are endemic to subtropical areas of southeast Asia (12). The animals are easily bred in captivity. In addition, cultures of primary tupaia hepatocytes (PTH) can be prepared using well-established liver perfusion protocols.We previously described HBV infection of PTH (17); infection efficiency was too low, h...

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