Roles of the Envelope Proteins in the Amplification of Covalently Closed Circular DNA and Completion of Synthesis of the Plus-Strand DNA in Hepatitis B Virus

Lentz, Thomas B.; Loeb, Daniel D.

doi:10.1128/jvi.05373-11

Cited by 71 publications

(68 citation statements)

References 57 publications

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“…To analyze the potential effects of the HBc mutants on reverse transcription and CCC DNA synthesis inside the cells, we transfected HepG2 and Huh7 cells with HBV-replicating constructs harboring these NTD mutations. Two versions of these constructs were used, either with or without the expression of the viral envelope proteins, which are known to regulate CCC DNA formation (9,27,29). We found that in both cell lines, either with or without expression of the envelope proteins, the L95A and K96A mutants showed a core DNA (i.e., NC-associated DNA) pattern similar to that of the WT ( Fig.…”

Section: Resultsmentioning

confidence: 78%

See 1 more Smart Citation

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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Section: Resultsmentioning

confidence: 78%

“…The two alternative fates of mature NCs (i.e., envelopment versus CCC DNA amplification) are known to be regulated by the viral envelope proteins (9,(27)(28)(29). Since HBc forms the NC shell, it is also likely to play a key role in these processes (30,31).…”

mentioning

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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“…Similarly, ablation of HBsAg also increased intranuclear HBV cccDNA formation, although to a lesser extent (7). Detailed examination using large (L), middle (M), and small (S) envelope protein mutant constructs suggested that L and M proteins were mainly responsible for this phenomenon (28). In liver tissues from CHB patients, intrahepatic HBsAg levels showed an inverse correlation with serum HBsAg titers (29).…”

Section: Localization Of Hbcag In Clinical Hbv Infectionmentioning

confidence: 93%

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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“…Regarding viral factors, the envelope proteins, especially the largest one, L, negatively regulate CCC DNA formation (10,(12)(13)(14)(15). In addition, as NC uncoating and RC DNA release must precede CCC DNA formation, the maturation-associated NC destabilization, which we have reported recently (16), is likely a prerequisite for CCC DNA formation.…”

mentioning

confidence: 99%

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

Cui

Guo

2015

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. For reasons yet to be understood, HBV is apparently unable to make CCC DNA in normal mouse hepatocytes in the liver. We report here that HBV CCC DNA was formed efficiently in an immortalized mouse hepatocyte cell line, AML12HBV10, and this is associated with destabilization of mature NCs in these cells. These results suggest that destabilization of mature HBV NCs in AML12HBV10 cells facilitates efficient NC uncoating and subsequent CCC DNA formation. They further implicate NC uncoating as an important step in CCC DNA formation that is subject to host regulation and potentially a critical determinant of host range and/or cell tropism of HBV. IMPORTANCE Persistent infection by hepatitis B virus (HBV), afflicting hundreds of millions worldwide, is sustained by the episomal viral covalently closed circular (CCC)DNA in the nuclei of infected hepatocytes. CCC DNA is converted from the viral genomic (precursor) DNA contained in cytoplasmic viral nucleocapsids. The conversion process remains ill defined, but host cell factors are thought to play an essential role. In particular, HBV fails to make CCC DNA in normal mouse hepatocytes despite the presence of large amounts of nucleocapsids containing the precursor viral DNA. We have found that in an immortalized mouse hepatocyte cell line, HBV is able to make abundant amounts of CCC DNA. This ability correlates with increased instability of viral nucleocapsids in these cells, which likely facilitates nucleocapsid disassembly (uncoating) to release the genomic DNA for conversion to CCC DNA. Our studies have thus revealed a novel mechanism of controlling viral persistence via regulating nucleocapsid disassembly. Hepatitis B virus (HBV) has infected approximately 2 billion people worldwide, with ca. 350 million of those 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). HBV genome replication starts with the assembly of a replication-competent but immature nucleocapsid (NC) by multiple copies of a single viral protein, the HBV core (HBc) protein, incorporating pgRNA and the viral reverse transcriptase (RT). Within the immature NCs, RT converts pgRNA first to a single-stranded (SS) (minus-strand) DNA and then to the DS RC DNA, and the immature NCs ar...

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Roles of the Envelope Proteins in the Amplification of Covalently Closed Circular DNA and Completion of Synthesis of the Plus-Strand DNA in Hepatitis B Virus

Cited by 71 publications

References 57 publications

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

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

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

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

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