Hepatitis B Virus Covalently Closed Circular DNA Formation in Immortalized Mouse Hepatocytes Associated with Nucleocapsid Destabilization

Cui, Xing; Guo, Ju Tao; Hu, Jianming

doi:10.1128/jvi.01261-15

Cited by 50 publications

(60 citation statements)

References 37 publications

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“…As we reported recently (41), HBV NCs are porous in AML12HBV10 cells and are unable to protect their RC DNA content from exogenous nuclease, and this is associated with enhanced CCC DNA formation in the nucleus and exposure of viral DNA in the cytoplasm. The failure of AML12HBV10 cells to maintain HBV gene expression and replication, coupled with the destabilization of viral NCs in these cells, prompted us to hypothesize that NC destabilization may actually be responsible for the shutdown of viral replication in AML12HBV10 cells.…”

Section: Resultsmentioning

confidence: 67%

“…For reasons yet to be clarified, mature HBV NCs become hyperdestabilized in the cytoplasm of AML12HBV10 cells (41), AML12HBV10 cells were induced to replicate HBV in Tet-free medium for 6 days in the presence of 0.1% DMSO (mock) (2nd row) or the presence of ETV (100 nM) (3rd row), AT-Br (20 M) (4th row), or BX795 (1 M) (bottom row). The cells were then harvested for immunofluorescence assay to determine IRF3 (green) subcellular localization using confocal microscopy.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, we have found that AML12HBV10 cells, in contrast to normal mouse hepatocytes in vivo, which are unable to support HBV CCC DNA formation despite accumulating abundant RC DNA (40), could support efficient HBV CCC DNA formation, which was likely facilitated by the rapid and efficient uncoating of the viral NCs to expose the genomic DNA for CCC DNA conversion in these cells (41). Here, we report that the increased exposure of RC DNA in AML12HBV10 cells led to the triggering of an innate immune response that was dependent on viral DNA and host DNA sensing and signaling mechanisms and was able to modulate viral gene expression and replication.…”

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confidence: 99%

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Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes

Cui

Clark

Liu

et al. 2016

J Virol

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Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. A prerequisite for CCC DNA formation is the uncoating (disassembly) of NCs to expose their RC DNA content for conversion to CCC DNA. We report here that in an immortalized mouse hepatocyte cell line, AML12HBV10, in which RC DNA exposure is enhanced, the exposed viral DNA could trigger an innate immune response that was able to modulate viral gene expression and replication. When viral gene expression and replication were low, the innate response initially stimulated these processes but subsequently acted to shut off viral gene expression and replication after they reached peak levels. Inhibition of viral DNA synthesis or cellular DNA sensing and innate immune signaling diminished the innate response. These results indicate that HBV DNA, when exposed in the host cell cytoplasm, can function to trigger an innate immune response that, in turn, modulates viral gene expression and replication. IMPORTANCE Chronic infection by hepatitis B virus (HBV) afflicts hundreds of millions worldwide and is sustained by the episomal covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes.Release of viral genomic DNA from cytoplasmic nucleocapsids (NCs) (NC disassembly or uncoating) is a prerequisite for its conversion to CCC DNA, which can also potentially expose the viral DNA to host DNA sensors and trigger an innate immune response. We have found that in an immortalized mouse hepatocyte cell line in which efficient CCC DNA formation was associated with enhanced exposure of nucleocapsid-associated DNA, the exposed viral DNA indeed triggered host cytoplasmic DNA sensing and an innate immune response that was able to modulate HBV gene expression and replication. Thus, HBV can, under select conditions, be recognized by the host innate immune response through exposed viral DNA, which may be exploited therapeutically to clear viral persistence. Hepatitis B virus (HBV) has infected approximately 2 billion people worldwide, with 350 million of them becoming chronically infected (1). Annually, 1 million fatalities are attributed to acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) caused by HBV. HBV is a small, enveloped DNA virus that contains a 3.2-kb, partially double-stranded (DS), relaxed circular (RC) DNA genome and replicates via an RNA intermediate, the pregenomic RNA (pgRNA). Upon infection, HBV RC DNA is converted to covalently closed circular (CCC) DNA in the nuclei of infected human hepatocytes, which serves as the transcriptional template for all viral RNAs, incl...

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Section: Resultsmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes

Cui

Clark

Liu

et al. 2016

J Virol

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“…It remains also possible that the thermodynamic stability of NCs during maturation is actually unaltered by these mutations but a barrier to active uncoating (NC disassembly) is lowered: e.g., by prematurely exposing a putative uncoating signal as a result of the mutations. Interestingly, the properties of the I126A mutant mature NCs are phenocopied by WT mature NCs formed in an immortalized mouse hepatocyte cell line, AML12HBV10, which also show a lack of accumulation of RC DNA and loss of integrity of mature NCs, accompanied by high levels of PF-RC DNA and CCC DNA (54). This suggests that NC integrity and uncoating are also subject to host regulation.…”

Section: Discussionmentioning

confidence: 99%

“…This suggests that NC integrity and uncoating are also subject to host regulation. As normal mouse hepatocytes are unable to form CCC DNA or significant amounts of PF-RC DNA (54,55), possibly due to a deficiency in NC uncoating (54), and are resistant to HBV infection, even after receptor reconstitution (56,57), these results further implicate NC disassembly or uncoating as a critical step in determining HBV species tropism.…”

Section: Discussionmentioning

confidence: 99%

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation

Cui

Luckenbaugh

Bruss

et al. 2015

J Virol

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Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. IMPORTANCEHepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.T he human pathogen hepatitis B virus (HBV) belongs to the family of Hepadnaviridae, a group of small, hepatotropic DNA viruses that also include closely related animal viruses, such as the duck hepatitis B virus (DHBV) (1). Hepadnaviruses contain a small (ca. 3-kb), partially double-stranded (ds-), relaxed circular (RC) DNA genome enclosed within an icosahedral capsid that is, in turn, formed by multiple copies (240 or 180) of ...

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In vivo models of hepatitis B and C virus infection

et al. 2016

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Globally, more than 500 million individuals are chronically infected with hepatitis B (HBV), delta (HDV), and/or C (HCV) viruses, which can result in severe liver disease. Mechanistic studies of viral persistence and pathogenesis have been hampered by the scarcity of animal models. The limited species and cellular host range of HBV, HDV, and HCV, which robustly infect only humans and chimpanzees, have posed challenges for creating such animal models. In this review, we will discuss the barriers to interspecies transmission and the progress that has been made in our understanding of the HBV, HDV, and HCV life cycles. Additionally, we will highlight a variety of approaches that overcome these barriers and thus facilitate in vivo studies of these hepatotropic viruses.

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Hepatitis B Virus Covalently Closed Circular DNA Formation in Immortalized Mouse Hepatocytes Associated with Nucleocapsid Destabilization

Cited by 50 publications

References 37 publications

Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes

Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation

In vivo models of hepatitis B and C virus infection

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