Hepatitis B virus (HBV), the causative agent of chronic hepatitis B and prototypic hepadnavirus, is a small DNA virus that replicates by protein-primed reverse transcription. The product is a 3-kb relaxed circular DNA (RC-DNA) in which one strand is linked to the viral polymerase (P protein) through a tyrosyl-DNA phosphodiester bond. Upon infection, the incoming RC-DNA is converted into covalently closed circular (ccc) DNA, which serves as a viral persistence reservoir that is refractory to current anti-HBV treatments. The mechanism of cccDNA formation is unknown, but the release of P protein is one mandatory step. Structural similarities between RC-DNA and cellular topoisomerase-DNA adducts and their known repair by tyrosyl-DNA-phosphodiesterase (TDP) 1 or TDP2 suggested that HBV may usurp these enzymes for its own purpose. Here we demonstrate that human and chicken TDP2, but only the yeast ortholog of TDP1, can specifically cleave the Tyr-DNA bond in virus-adapted model substrates and release P protein from authentic HBV and duck HBV (DHBV) RC-DNA in vitro, without prior proteolysis of the large P proteins. Consistent with TPD2's having a physiological role in cccDNA formation, RNAi-mediated TDP2 depletion in human cells significantly slowed the conversion of RC-DNA to cccDNA. Ectopic TDP2 expression in the same cells restored faster conversion kinetics. These data strongly suggest that TDP2 is a first, although likely not the only, host DNA-repair factor involved in HBV cccDNA biogenesis. In addition to establishing a functional link between hepadnaviruses and DNA repair, our results open new prospects for directly targeting HBV persistence.hepatitis B virus persistence | TDP substrate specificity | virus-DNA repair interface M ore than 250 million people worldwide are chronically infected with hepatitis B virus (HBV) (1) and are at a highly increased risk for developing end-stage liver disease (2). Current treatments with IFN-α or nucleos(t)ide analogs (NAs) are only partially effective (3, 4). Importantly, they do not directly target the viral persistence reservoir, an episomal covalently closed circular (ccc) DNA form of the viral genome that serves as template for all viral transcripts; hence a few cccDNA molecules present in the liver can reactivate full viral replication. Eliminating infection thus will require the elimination of cccDNA. cccDNA is generated, upon infection, from the viral polymerase (P protein)-linked relaxed circular (RC) DNA present in incoming virions (Fig. 1A). The mechanism by which RC-DNA is converted to cccDNA is poorly understood, but it must involve multiple steps (see below). Because the ∼3-kb genomes of HBV and the other hepadnaviruses [e.g., from ducks (DHBV)] are too small to encode all the activities required for this conversion, these activities must be provided by the host cell.RC-DNA from all hepadnaviruses bears several molecular peculiarities that result from its generation by protein-primed reverse transcription (for reviews, see refs. 5 and 6). The pregenomic RNA (p...

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.

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...

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