Functional Surfaces of the Hepatitis B Virus Capsid

ABSTRACT The hepatitis B virus (HBV) core protein (CP) forms the shell of an icosahedral nucleocapsid. In a former work, we identified 11 amino acid residues of CP exposed on the capsid surface by an alanine mutation scan as being important for capsid envelopment. We now introduced several other amino acids at six of these positions and found that almost all 27 tested point mutations at S17, K96, and I126 reproduced the phenotype of the alanine mutation (with only two exceptions): the formation of nucleocapsids and of the viral DNA genome was wild type, but capsid envelopment and virion release were strongly inhibited. This indicates that these side chains have a very specific function during nucleocapsid envelopment. We also identified several CP point mutations (e.g., F122V/S/Y and R127D/G) allowing the formation of capsids but preventing the packaging of pregenomic RNA. The envelopment of such mutant capsids was blocked. Apparently, these CP mutations hampered the recognition/packaging of the pregenome-P-protein complex by CP, a process which is still barely understood, and the mutant capsids devoid of HBV-specific nucleic acid did not express the capsid maturation signal required for envelopment.

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Hepatitis B Virus Replication and Release Are Independent of Core Lysine Ubiquitination

Journal of Virology ◽

10.1128/jvi.02644-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 4923-4933 ◽

Cited By ~ 15

Author(s):

Mayra L. Garcia ◽

Rushelle Byfield ◽

Michael D. Robek

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Ubiquitin Ligase ◽

Core Protein ◽

Structural Proteins ◽

Hbv Replication ◽

Virion Release ◽

B Virus ◽

Lysine Residues ◽

Ubiquitin Conjugation

ABSTRACT Ubiquitin conjugation to lysine residues regulates a variety of protein functions, including endosomal trafficking and degradation. While ubiquitin plays an important role in the release of many viruses, the requirement for direct ubiquitin conjugation to viral structural proteins is less well understood. Some viral structural proteins require ubiquitin ligase activity, but not ubiquitin conjugation, for efficient release. Recent evidence has shown that, like other viruses, hepatitis B virus (HBV) requires a ubiquitin ligase for release from the infected cell. The HBV core protein contains two lysine residues (K7 and K96), and K96 has been suggested to function as a potential ubiquitin acceptor site based on the fact that previous studies have shown that mutation of this amino acid to alanine blocks HBV release. We therefore reexamined the potential connection between core lysine ubiquitination and HBV replication, protein trafficking, and virion release. In contrast to alanine substitution, we found that mutation of K96 to arginine, which compared to alanine is more conserved but also cannot mediate ubiquitin conjugation, does not affect either virus replication or virion release. We also found that the core lysine mutants display wild-type sensitivity to the antiviral activity of interferon, which demonstrates that ubiquitination of core lysines does not mediate the interferon-induced disruption of HBV capsids. However, mutation of K96 to arginine alters the nuclear-cytoplasmic distribution of core, leading to an accumulation in the nucleolus. In summary, these studies demonstrate that although ubiquitin may regulate the HBV replication cycle, these mechanisms function independently of direct lysine ubiquitination of core protein.

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Influence of a Putative Intermolecular Interaction between Core and the Pre-S1 Domain of the Large Envelope Protein on Hepatitis B Virus Secretion

Journal of Virology ◽

10.1128/jvi.76.13.6510-6517.2002 ◽

2002 ◽

Vol 76 (13) ◽

pp. 6510-6517 ◽

Cited By ~ 33

Author(s):

Sophie Le Pogam ◽

Chiaho Shih

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Wild Type ◽

The Core ◽

Virion Release ◽

Naturally Occurring ◽

B Virus ◽

Kinetics Of ◽

Viral Reverse Transcription

ABSTRACT Virion release of hepatitis B virus (HBV) from hepatocytes is a tightly regulated event. It is a dogma that only the mature HBV genome is preferentially allowed to export from the intracellular compartment (J. Summers and W. S. Mason, Cell 29:403-415, 1982). Recently, an “immature secretion” phenotype of a highly frequent naturally occurring HBV variant containing a leucine residue at amino acid 97 of the core protein was identified. Unlike wild-type HBV, this variant secretes almost equal amounts of mature and immature genomes. This phenomenon is not caused by any instability of core particles or by any deficiency in viral reverse transcription (T. T. Yuan, P. C. Tai, and C. Shih, J. Virol. 73:10122-10128, 1999). In this study, our kinetic analysis of virion secretion of the mutant F97L (phenylalanine to leucine) indicates that the secretion of its immature genome does not occur earlier than that of its mature genome. In addition, the secretion kinetics of the mature genomes are comparable between the wild-type HBV and the mutant F97L. Therefore, the immature secretion phenomenon of mutant F97L is not caused by premature secretion or more efficient secretion. Previously, we hypothesized that the immature secretion phenotype is probably caused by the aberrant interaction between its mutant core and wild-type envelope proteins. Here, we further demonstrated that a pre-S1 envelope mutation at position 119, changing an alanine (A) to a phenylalanine (F), can offset the immature secretion phenotype of the mutant I97L (isoleucine to leucine) and successfully restore the wild-type-like selective export of the mature genome of the double mutant pre-S1-A119F/core-I97L.

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Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis

PLoS Pathogens ◽

10.1371/journal.ppat.1010057 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1010057

Author(s):

Hui Liu ◽

Junjun Cheng ◽

Usha Viswanathan ◽

Jinhong Chang ◽

Fengmin Lu ◽

...

Keyword(s):

Amino Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Amino Acid Residues ◽

Viral Dna ◽

Viral Dna Replication ◽

Dimer Interface ◽

Rich Domain ◽

B Virus

The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic “HAP” pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically “normal” capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.

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The Hepatitis B Virus Nucleocapsid—Dynamic Compartment for Infectious Virus Production and New Antiviral Target

Biomedicines ◽

10.3390/biomedicines9111577 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1577

Author(s):

Matthias Niklasch ◽

Peter Zimmermann ◽

Michael Nassal

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcription ◽

Therapeutic Potential ◽

Core Protein ◽

Virus Production ◽

P Protein ◽

Capsid Shell ◽

B Virus ◽

Stable Genome

Hepatitis B virus (HBV) is a small enveloped DNA virus which replicates its tiny 3.2 kb genome by reverse transcription inside an icosahedral nucleocapsid, formed by a single ~180 amino acid capsid, or core, protein (Cp). HBV causes chronic hepatitis B (CHB), a severe liver disease responsible for nearly a million deaths each year. Most of HBV’s only seven primary gene products are multifunctional. Though less obvious than for the multi-domain polymerase, P protein, this is equally crucial for Cp with its multiple roles in the viral life-cycle. Cp provides a stable genome container during extracellular phases, allows for directed intracellular genome transport and timely release from the capsid, and subsequent assembly of new nucleocapsids around P protein and the pregenomic (pg) RNA, forming a distinct compartment for reverse transcription. These opposing features are enabled by dynamic post-transcriptional modifications of Cp which result in dynamic structural alterations. Their perturbation by capsid assembly modulators (CAMs) is a promising new antiviral concept. CAMs inappropriately accelerate assembly and/or distort the capsid shell. We summarize the functional, biochemical, and structural dynamics of Cp, and discuss the therapeutic potential of CAMs based on clinical data. Presently, CAMs appear as a valuable addition but not a substitute for existing therapies. However, as part of rational combination therapies CAMs may bring the ambitious goal of a cure for CHB closer to reality.

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In vivo Suppression der Hepatitis B Virus Expression und Replikation durch das Hepatitis C Core Protein in einem hydrodynamischen Transfektionsmodell

Zeitschrift für Gastroenterologie ◽

10.1055/s-2005-919967 ◽

2005 ◽

Vol 43 (05) ◽

Author(s):

C Gehrke ◽

C Klein ◽

N Woller ◽

MP Manns ◽

C Trautwein

Keyword(s):

Hepatitis C ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

B Virus ◽

Virus Expression

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RBM24 stabilizes hepatitis B virus pregenomic RNA but inhibits core protein translation by targeting the terminal redundancy sequence

Emerging Microbes & Infections ◽

10.1038/s41426-018-0091-4 ◽

2018 ◽

Vol 7 (1) ◽

pp. 1-14 ◽

Cited By ~ 7

Author(s):

Yongxuan Yao ◽

Bo Yang ◽

Huang Cao ◽

Kaitao Zhao ◽

Yifei Yuan ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Protein Translation ◽

B Virus

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Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

Journal of General Virology ◽

10.1099/vir.0.83149-0 ◽

2007 ◽

Vol 88 (12) ◽

pp. 3270-3274 ◽

Cited By ~ 27

Author(s):

Marianne Bonvin ◽

Jobst Greeve

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Point Mutations ◽

Alternative Mechanism ◽

Amino Terminal ◽

Hbv Replication ◽

B Virus ◽

Carboxy Terminal ◽

Deaminase Domain

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

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