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

Köck, Josef; Rösler, Christine; Zhang, Jingjing; Blum, Hubert E.; Nassal, Michael; Thoma, Christian

doi:10.1371/journal.ppat.1001082

Cited by 131 publications

(153 citation statements)

References 58 publications

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“…It should be noted that double-stranded linear DNA (dslDNA) was also present, since SpeI digestion resulted in a 1.2-kb band in addition to 3.2-kb species (lane 8). A heat resistance assay also confirmed the cccDNA form in protein-free DNA from liver biopsies when samples were heated from 75°C to 95°C ( Figure 3B, lanes 2-5), while PF-rcDNA from HepAD38 cells (lanes 6-9) and core particle DNA from a liver biopsy (lanes [11][12][13][14] were readily denatured. In addition, topoisomerase I treatment transformed the cccDNA into the relaxed form, as shown in Figure 3C (lanes 1 and 2), while PF-rcDNA from HepAD38 cells showed no mobility change (lanes 4 and 5).…”

Section: Resultsmentioning

confidence: 67%

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

Zhang¹,

Lu²,

Zheng³

et al. 2016

Journal of Clinical Investigation

107

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Section: Resultsmentioning

confidence: 67%

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

Zhang¹,

Lu²,

Zheng³

et al. 2016

Journal of Clinical Investigation

107

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“…Transfected cells were lysed in Nonidet P-40 lysis buffer, and the nuclei were separated by low-speed centrifugation as previously described (39). Nuclear DNAs were prepared using the QiaAmp Blood Mini Kit (Qiagen) according to the manufacturer´s tissue protocol for subsequent Southern blotting.…”

Section: Methodsmentioning

confidence: 99%

“…Southern blotting was performed as previously described (39). 32 P-labeled DNA probes were obtained by random priming using cloned full-length DHBV or HBV genomes as templates.…”

Section: Methodsmentioning

confidence: 99%

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

Königer

Wingert

Marsmann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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210

193

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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...

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“…To note, cccDNA can be derived not only from the up-taken virions but also by the synthetized nucleocapsids that are transported into the nucleus without being secreted into the bloodstream. This mechanism represents the basis of the accumulation and maintenance of cccDNA pool (19,20).…”

Section: Hbv Replication Cyclementioning

confidence: 99%

Untitled

2017

Istanbul Med J

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Hepatitis B virus (HBV) is a small-enveloped DNA virus that belongs to the Hepadnaviridae family, representing the second greatest cause of chronic viral hepatitis worldwide (1). Despite decades of vaccination, HBV infection is still a major global burden, with 257 million persons chronically infected and approximately 880,000 deaths per year (2). Chronic hepatitis B (CHB) is the result of an acute, unresolved infection that induces an immune-mediated liver damage followed by fibrotic tissue deposition leading overtime to a complete alteration of the hepatic architecture toward cirrhosis and its complications, such as liver failure and hepatocellular carcinoma (HCC) (3, 4).Remarkable advances have been made in the setting of CHB treatment (5). In particular, the development of new molecular biology techniques in association with an ever deeper understanding of the different phases of HBV infection and primarily the introduction of nucleos(t)ide analogs (NAs) are the main features that may lead to the virological cure in the near term and the functional cure in the long-term follow-up (6).This review discusses the current available drugs and treatment guidelines for CHB therapy focusing on telbivudine (LtD), a thymidine analog that has demonstrated unique features that make the abovementioned drug still valid even in the era of new powerful anti-viral agents. HBV Virology and Replication CycleHBV structure Hepatitis B virus structure consists of an outer envelope made of three viral surface proteins named preS1 (large), preS2 (middle), and S (small), which correspond to the serologic HBsantigen (HBsAg), and an inner nucleocapsid formed by core proteins, which correspond to HBcore-antigen (HBcAg) (7). Within the nucleocapsid is present the full-length HBV genome as a partially double-stranded relaxed circular (rc) DNA linked to the viral polymerase protein by a phosphotyrosine bond (8). The genome has an extremely compact organization displaying four partially overlapped major open reading frames (ORFs): the preS/S that encodes the three viral surface proteins, the pre-core/core that encodes both the nucleocapsid core protein and the non-structural core protein known as the e-antigen (HBeAg); the polymerase ORF that encodes the viral polymerase; and the X ORF that encodes the regulatory X protein that is essential for viral replication (9). Chronic Hepatitis B Treatment: Current Perspectives on TelbivudineHepatitis B virus (HBV) is the primary cause of chronic viral hepatitis worldwide. Currently, there are 5 oral nucleos(t)ide analogs (NAs) approved for chronic hepatitis B (CHB) treatment and include lamivudine, adefovir, telbivudine (LtD), entecavir (ETV), and tenofovir disoproxil fumarate (TDF). ETV and TDF are those with higher anti-viral efficacy and lower resistance rates, whereas LtD shows similar efficacy but has a higher risk for viral resistance in long-term monotherapy. However, LtD carries several advantages that must be considered and are worthy of discussion. Compared to other NAs, LtD showed a poten...

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

Cited by 131 publications

References 58 publications

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

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