Hepatitis B Virus Virology and Replication

2017

Viruses

Self Cite

239

245

Hepatitis B virus (HBV) is a para-retrovirus or retroid virus that contains a double-stranded DNA genome and replicates this DNA via reverse transcription of a RNA pregenome. Viral reverse transcription takes place within a capsid upon packaging of the RNA and the viral reverse transcriptase. A major characteristic of HBV replication is the selection of capsids containing the double-stranded DNA, but not those containing the RNA or the single-stranded DNA replication intermediate, for envelopment during virion secretion. The complete HBV virion particles thus contain an outer envelope, studded with viral envelope proteins, that encloses the capsid, which, in turn, encapsidates the double-stranded DNA genome. Furthermore, HBV morphogenesis is characterized by the release of subviral particles that are several orders of magnitude more abundant than the complete virions. One class of subviral particles are the classical surface antigen particles (Australian antigen) that contain only the viral envelope proteins, whereas the more recently discovered genome-free (empty) virions contain both the envelope and capsid but no genome. In addition, recent evidence suggests that low levels of RNA-containing particles may be released, after all. We will summarize what is currently known about how the complete and incomplete HBV particles are assembled. We will discuss briefly the functions of the subviral particles, which remain largely unknown. Finally, we will explore the utility of the subviral particles, particularly, the potential of empty virions and putative RNA virions as diagnostic markers and the potential of empty virons as a vaccine candidate.

Section: Introductionmentioning

confidence: 99%

Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application

2017

Viruses

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239

245

“…HBV is an enveloped virus with an inner capsid shell which, in turn, encloses a small (3.2-kbp) DNA genome. As a member of the Hepadnaviridae family, which also includes related animal viruses such as the duck hepatitis B virus (DHBV), HBV replicates its DNA genome via reverse transcription of an RNA intermediate called pregenomic RNA (pgRNA) (3)(4)(5). HBV assembly begins with the formation of a nucleocapsid (NC) that packages specifically a copy of pgRNA, in complex with the virally encoded reverse transcriptase (RT) protein (6,7).…”

mentioning

confidence: 99%

Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation

Ludgate

Luckenbaugh

et al. 2016

J Virol

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126

Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors. IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancerworldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.

“…epadnaviruses are small DNA viruses with an icosahedral capsid and an outer envelope (1). This family of viruses includes the human hepatitis B virus (HBV) and the widely used animal model virus, the duck hepatitis B virus (DHBV).…”

mentioning

confidence: 99%

“…Hepadnavirus assembly starts with the packaging of the viral pregenomic RNA (pgRNA), a precursor to the genomic DNA, into a replication-competent nucleocapsid (NC). Packaging of pgRNA into NCs is dependent on the viral polymerase protein, a specialized reverse transcriptase (RT), which specifically recognizes the pgRNA and is packaged together with the pgRNA as a ribonucleoprotein complex by the assembling capsid protein subunits (1). Following NC assembly, the RT protein converts the pgRNA first to the single-stranded (minusstrand) DNA and then to a partially double-stranded, relaxed circular (RC) DNA (3).…”

mentioning

confidence: 99%

“…Both the pgRNA-and single-stranded DNA (ssDNA)-containing NCs are considered immature, as they are not enveloped for secretion as virions extracellularly. Only the dsDNA-containing NC, considered mature, is selectively recognized by the viral envelope or surface proteins for envelopment and secretion as complete virions (1,4). This requirement of dsDNA synthesis for NC envelopment and virion formation is thus a major characteristic of hepadnaviruses (DNA viruses) that distinguishes them from conventional retroviruses (RNA viruses).…”

mentioning

confidence: 99%

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Capsid Phosphorylation State and Hepadnavirus Virion Secretion

Ning

Basagoudanavar

et al. 2017

J Virol

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The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging into immature nucleocapsids (NCs) and the early stage of viral DNA synthesis. For the avian hepadnavirus duck hepatitis B virus (DHBV), CTD is dephosphorylated subsequently to facilitate the late stage of viral DNA synthesis and to stabilize NCs containing mature viral DNA. The role of CTD phosphorylation in virion secretion, if any, has remained unclear. Here, the CTD from the human hepatitis B virus (HBV) was found to be dephosphorylated in association with NC maturation and secretion of DNA-containing virions, as in DHBV. In contrast, the CTD in empty HBV virions (i.e., enveloped capsids with no RNA or DNA) was found to be phosphorylated. The potential role of CTD dephosphorylation in virion secretion was analyzed through mutagenesis. For secretion of empty HBV virions, which is independent of either viral RNA packaging or DNA synthesis, multiple substitutions in the CTD to mimic either phosphorylation or dephosphorylation showed little detrimental effect. Similarly, phospho-mimetic substitutions in the DHBV CTD did not block the secretion of DNA-containing virions. These results indicate that CTD dephosphorylation, though associated with NC maturation in both HBV and DHBV, is not essential for the subsequent NC-envelope interaction to secrete DNA-containing virions, and the CTD state of phosphorylation also does not play an essential role in the interaction between empty capsids and the envelope for secretion of empty virions.IMPORTANCE The phosphorylation state of the C-terminal domain (CTD) of hepatitis B virus (HBV) core or capsid protein is highly dynamic and plays multiple roles in the viral life cycle. To study the potential role of the state of phosphorylation of CTD in virion secretion, we have analyzed the CTD phosphorylation state in complete (containing the genomic DNA) versus empty (genome-free) HBV virions. Whereas CTD is unphosphorylated in complete virions, it is phosphorylated in empty virions. Mutational analyses indicate that neither phosphorylation nor dephosphorylation of CTD is required for virion secretion. These results demonstrate that while CTD dephosphorylation is associated with HBV DNA synthesis, the CTD state of phosphorylation may not regulate virion secretion.