Core virus b

Notably, in some Dane particles the envelope was only loosely associated with the core 30 , this may be a function of the distribution of the different forms of the HBsAg.

The capsid does not firmly constrain the distribution of HBsAg units in the envelope but provides a generalized template for packing A composite cryo-EM reconstruction of a dane particle. Views are cross-sections left , and two cut aways b X-ray crystal structure of recombinant capsid 36 docked into the cryo-EM density map of the virion capsid left.

The tips of the core spikes are in close apposition but do not penetrate the envelope. Additional details and cartoon of interpretation right. The surface protein projections are ascribed to HBsAg and are designated as large L , medium M , and small S arbitrarily. These figures reproduced with permission from Dryden et al. The basic soluble unit of HBcAg is a dimer Figure 2a. A HBcAg monomer is residues long for in vitro studies we refer to it as core protein or Cp This domain is located inside the capsid and has been identified to be intrinsically disordered based on sequence and Cryo-EM studies The assembly domain alone is sufficient for assembly of morphologically regular empty capsids Helices 1, 2 and 5 are part the chassis of the capsid.

A tight proline-rich loop connects helix 5 to the CTD Holding together a dimer, helices 3 and 4 from one half-dimer form a 4-helix motif with corresponding helices from the other half-dimer.

This motif is flanked by salt bridges and hydrogen bonds that stabilize the hydrophobic interface. Helix 4 is kinked in the middle and is sometimes referred to as helices 4a and 4b. A disulfide bond can form over time between highly conserved C61 residues in helix 3, which contributes to the dimer interface 38 — X-ray crystal structures of the assembly domain. The dimer interface comprises of a four-helix bundle created by two helices from each monomer.

The free dimer is less compact than the dimer in the context of a capsid c An HBeAg dimer. Dimers in the context of capsids are more compact than free dimers in solution Figure 2b 36 , Crystal structures of an assembly-incompetent mutant YA of HBcAg 41 — 43 show substantial variability, mainly at the spike tips and the C-termini.

This variability in structure is thought to be indicative of the variety in functional roles for HBcAg in the viral life cycle. For example, an oxidized C61—C61 dimer results in weaker dimer-dimer interactions and slower capsid assembly rates in comparison to the reduced form 38 ; thus, the oxidized dimer favors a conformation or conformations that are unfavorable for capsid assembly.

While the cytoplasm where capsid assembly takes place during an infection is a reducing environment, the proximity of the C61 residues is high may be sufficient to overcome the reducing potential.

Data suggest that structural dynamics that allow HBcAg to transition between these states are an important key to its function 38 , 41 , 44 , HBeAg is a secreted protein expressed by every member of the Hepadnaviridae family though its expression is not required to maintain infection. The remaining 10 residues upstream of HBcAg including a Cys at position This C-7 forms an intramolecular disulfide with C61 to stabilize the structure HBeAg is thought to have an immune-modulatory role that allows for humoral and cell-mediated immune evasion 49 , Paradoxically, loss of e antigen is correlated with clearance of acute HBV infection and establishment of chronic infection In a similar vein, loss of HBeAg in culture correlates with increased HBV expression while in humans it correlates with decreased viremia While oxidized HBeAg does not assemble into capsids, sedimentation analysis and electron microscopy showed that reduced HBeAg dimers could 53 , Thus, it is possible that both HBeAg- and HBcAg-associated dimer conformations may be accessible to both proteins in solution.

Assembly studies with reduced HBeAg dimers indicate that their capsid-like products are less regular than HBcAg-assembled capsids This would suggest that co-assembly of HBcAg with HBeAg would have a negative impact on regular capsid assembly, both from a kinetic and thermodynamic point of view. Thus, the tip of the spike must have evolved to support both parallel and antiparallel interaction.

This gross change was stabilized by the disulfide from C-7 in the peptide to C61 of helix 3 one per monomer , which replaces the HBcAg C61—C61 disulfide The structure is also stabilized by hydrophobic contacts between the peptide and helix 3 and 4. The peptide covers some of the exposed hydrophobic surface at the base of helix 3, creating a new interface HBcAg can self-assemble to form the icosahedral virus capsid.

Icosahedra are comprised of 60 asymmetric units. The 4-helix motifs from the dimers are oriented perpendicular to the surface of the capsid, forming the spikes that punctuate the capsid. By convention, A monomers form the fivefold vertices and two sets of B, C and D monomers form the quasi-sixfold vertices Figure 2d.

Large holes fenestrate the capsid surface and provide a means for nucleotides and other small molecules to diffuse in and out of the particle.

This groove is filled by the helix-turn-extended structure of an incoming subunit A peculiar pocket formed at this interface is the basis of assembly-directed antivirals, which will be discussed later in this review.

The HBcAg capsid is itself highly immunogenic and reported to induce both B- and T-cell responses 64 , though these responses are not protective against HBV infection. Peptide mapping studies identified the spike tip, around residue 80, as the major epitope 56 , 65 In a series of cryo-EM studies antibodies to capsids were shown to bind not only to a linear epitope on the spike tip but to conformational epitopes on the sides of the spike and its base 35 , 66 — Other epitopes have been observed at interdimer contacts 65 , Because of its immunogenicity, HBV capsids have been used as a carrier for epitopes 69 , The residue or 36 residue, depending on genotype arginine-rich HBcAg CTD Figure 3a is localized to the interior of the capsid Figure 3b , though it can transiently be exposed on the capsid exterior 37 , 73 — The CTD can be considered a sequence of four arginine-rich repeats Figure 3.

The CTD has 7 conserved serines and a threonine which can be phosphorylated , , 79 , Figure 3a. These kinases may act on HBcAg prior to assembly or in the context of capsid. One or more types of kinase also gets packaged in cores, with strong evidence supporting identification as Cdk2 Phosphorylation of S, S, and S is critical for packaging RNA Figure 3a — substitution of these residues with alanine to mimic the unphosphorylated state suppresses pgRNA packaging 76 , 79 — The density in color corresponds to the CTD based on the fitting of an X-ray crystal structure of a Cp capsid in the reconstruction.

These figures reproduced with permission from Selzer et al. However, the affinity for dimer is fold stronger suggesting a thermodynamic basis for stalling assembly; SRPK also is large enough that it would occlude assembly of bound dimers. Thus, hypothetically SRPK can acts as a non-canonical chaperone that gates assembly. Transient exposure of CTDs in empty Cp capsids has also been documented by trypsin digestion studies Mass spectrometry of trypsin-digested empty capsids showed ready accessibility as far as residue Cryo-EM reconstructions of these products verified the cleavage and loss of CTD density underneath quasi-sixfold vertices Figure 3b There is a strong correlation between the positive charge of a viral capsid and its nucleic acid content 82 , 83 ; this correlation extends to HBV 84 — Decreasing the amount of positive charge in an HBV expression system, by truncating the CTD 85 or by replacing blocks of arginines with alanines 87 , decreases the amount of pgRNA encapsidated in cell culture expression of HBV.

Phosphorylation appears to modulate structural interactions between CTDs that affect capsid stability and RNA organization within the capsid. In empty Cp capsids, external exposure of CTDs is decreased and capsid stability is strengthened by an SE, SE, SE triple mutation, mimicking phosphorylation of the three critical serines In a virus expression system, the triple mutant assembles without packaging RNA The triple mutant phosphorylation mimic also modified the organization of packaged pgRNA in in vitro assembled particles In the phosphorylation-mimic mutant the RNA formed a more diffuse mesh-like network Figure 4b of thin and short stretches of electron density RNA containing capsids.

Icosahedral reconstructions of a wildtype Cp and b a phosphorylation mimic mutant with in vitro packaged pgRNA blue and gold respectively. The pgRNA in the former forms an icosahedral cage while the same in the latter is more mesh-like c An asymmetric reconstruction of an RNA-containing virion shows density for pgRNA gold , P protein red and other unassigned content.

These figures reproduced with permission from Wang et al. Though CTDs are ostensibly on the capsid interior, in empty capsids and DNA-filled capsids they are accessible to the capsid exterior 74 — These observations suggest that intracellular transport of free dimer, empty capsids, and mature capsids use different mechanisms. Efforts to clarify the specific NLS sites have been complicated by the fact that they may overlap. Furthermore, the same sequences in the CTD have been shown to act as nuclear export signals, presumably for Cp dimer; this has been suggested as a mechanism for export of viral RNA from the nucleus Reverse transcription reviewed in 23 , 93 , 94 is the target of most anti-HBV therapeutics now available.

The P protein has four domains, a terminal priming domain, a linker, a polymerase domain, and an RNaseH. The polymerase, though it is monomeric, has been modeled based on the HIV reverse transcriptase The RNAseH shares little sequence identity with other RNaseH proteins but enough to allow identification of active site residues and evaluate potential inhibitors Synthesis of the plus strand begins there, using the RNA as a primer.

After running out of template, the P complex with the growing plus strand switches template for the third time. Successful maturation of the HBV virion requires three template switches in which P and associated nucleic acids jump from one end of the template nucleic acid to the other. For these to anneal, the encapsidated P protein and pgRNA adopt a specific quaternary arrangement. RNA density was irregular but lined the interior surface of the capsid, similar to in vitro -assembled RNA-filled capsids Figure 4c.

The RNA density was not resolved enough to fit individual strands. Other unassigned internal density was observed and could be other packaged protein or could be an artifact arising from irregularity in the capsids and the difficulty of determining asymmetric orientation in an icosahedral particle.

The shell of the capsid as well as its interior surface impact reverse transcription. Mutation of conserved Val at the inter-dimer interface affects capsid assembly in a predictable way, changing capsid stability proportional to the change in buried surface Reverse transcription was also profoundly affected While first strand synthesis was proportional to the amount of pgRNA packaged, second strand synthesis was abolished , This suggests that the capsid is not just an inert carrier of genomic material and its structural properties affect seemingly unrelated processes.

The HBV Surface antigen has distinct roles in the virus lifecycle. Intracellularly, HBsAg binds cores, attenuating core transport to the nucleus and effectively regulating cccDNA copy number HBsAg is responsible for binding cores during assembly and receptor during infection.

The structure of HBsAg is known to low resolution only. At a basic level, HBsAg is a glycosylated integral membrane protein that has been localized to ER, pre-Golgi, and late endosomal membranes — M-HBsAg, which is not required for infection, lacks pre-S1. S-HBsAg is comprised of only the S domain which is composed of four trans-membrane helices The S domain forms disulfide crosslinked dimers, allowing formation of homo- and heterodimers. Subviral particles are heterogeneous in chemical makeup and structure.

Micrographs of subviral particles show serrations indicative of regular protrusions Figure 5a, b. However, spherical particles appear to be heterogeneous in size and geometry — they are not icosahedral — frustrating efforts to generate image reconstructions Filamentous particles also are heterogeneous but can be classified based on helical parameters eg.

Spikes in the reconstruction are similar in size to those in the micrograph. The volume of a given spike corresponds well with the expected volume for four S domains, suggesting the biologically relevant complex is a dimer of dimers HBsAg subviral particles. Note how some filaments vary in their diameter.

The scale bars correspond to nm. Residues from different monomers are in magenta and orange, respectively. Residues that are partially obscured by interdimer interfaces are highlighted in muted colors.

Panels a and b are reproduced with permission from Short et al. In most viruses, internal features of the envelope protein s interact with the virus core and external features mediate interaction between the virion and its receptor s.

In HBV, both of these activities are functions of the pre-S domains of L-HBsAg, a peptide that can change its localization over time and possibly in response to external triggers. In L-HBsAg pre-S1, which is myristoylated, and pre-S2 are largely cytoplasmic, based on proteolytic sensitivity and the absence of pre-S2 glycosylation. This places the pre-S domains where they can interact with newly matured HBV cores.

Conversely, in Dane particles the pre-S1 and pre-S2 domains are accessible to external proteases. Dane particles appear to first interact with host cells through a relatively weak electrostatic interaction to highly sulfated proteoglycan HSPG ; this interaction is a necessary precursor to infection , In vitro other anionic glycans, e.

Moreover, core proteins with point mutations at the wall of the HAP pocket, VA and VW, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Table 1. Data for 64 Hemodialysis Patients Were Unavailable a.

Open in a separate window. Figure 1. Figure 2. Figure 3. Figure 4. Discussion Occult hepatitis B infection is a major health concern globally, and a major cause of chronic liver diseases, cirrhosis, and infections transmitted by blood transfusion Acknowledgments This project was conducted as a research project with registration No at the Ahvaz Jundishapur university of medical sciences, Ahvaz, Iran. References 1. Mutation analysis of hepatitis B virus reverse transcriptase region among untreated chronically infected patients in Ahvaz city South-West of Iran.

Indian J Med Microbiol. The influence of human leukocyte antigen and IL gene polymorphisms on hepatitis B virus outcome. Hepat Mon. Epidemiology of occult hepatitis B infection among thalassemic, hemophilia, and hemodialysis patients. Occult hepatitis B among Iranian hepatitis C patients. J Res Med Sci. Viral deletions among healthy young Chinese adults with occult hepatitis B virus infection.

Virus Res. Hu KQ. Occult hepatitis B virus infection and its clinical implications. J Viral Hepat. J Hepatol. Zuckerman AJ. Effect of hepatitis B virus mutants on efficacy of vaccination. Enhancement of HBsAg detection in serum of patients with chronic liver disease following removal of circulating immune complexes.

High incidence of hepatitis B infections among chronic hepatitis cases of unknown aetiology. Surface gene mutations of hepatitis B virus among high-risk patients with occult hepatitis B virus infection. Diagn Microbiol Infect Dis. Occult hepatitis B virus infection among blood donors with antibodies to hepatitis B core antigen. Acta Med Iran. Occult hepatitis B virus infection in hemodialysis patients: a concept for consideration.

Ther Apher Dial. Post-transfusion occult hepatitis B OBI : a global challenge for blood recipients and health authorities. Hepatitis B virus genotypes and hepatitis B surface antigen mutations in family contacts of hepatitis B virus infected patients with occult hepatitis B virus infection.

J Gastroenterol Hepatol. Polymorphisms within Fas gene are not associated with occult hepatitis B virus infection: Polymorphisms within Fas gene in occult HBV infection. Chronic Hepatitis B Chronic hepatitis B has a variable and dynamic course.

Important Questions and Needs for Future Research How does HBV establish productive infection in vivo and what is the host response early during the infection? Footnotes Potential conflict of interest: Nothing to report. References 1. Ganem D, Schneider RJ. Hepadnaviridae and their replication.

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X-deficient woodchuck hepatitis virus mutants behave like attenuated viruses and induce protective immunity in vivo. J Clin Invest. Altered proteolysis and global gene expression in hepatitis B virus X transgenic mouse liver. The enigmatic X gene of hepatitis B virus.

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Phosphorylation of hepatitis B virus Cp at Ser87 facilitates core assembly. Biochem J. Kock J, Schilicht H-J. Analysis of the earliest steps of hepadnavirus replication genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity.

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