[The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016].

This review article is a continuation of the paper "Hepatitis B core particles as a universal display model: a structure-function basis for development" written by Pumpens P. and Grens E., ordered by Professor Lev Kisselev and published in FEBS Letters, 1999, 442, 1-6. The past 17 years have strengthened the paper's finding that the human hepatitis B virus core protein, along with other Hepadnaviridae family member core proteins, is a mysterious, multifunctional protein. The core gene of the Hepadnaviridae genome encodes five partially collinear proteins. The most important of these is the HBV core protein p21, or HBc. It can self-assemble by forming viral HBc particles, but also plays a crucial role in the regulation of viral replication. Since 1986, the HBc protein has been one of the first and the most successful tools of the virus-like particle (VLP) technology. Later, the woodchuck hepatitis virus core protein (WHc) was also used as a VLP carrier. The Hepadnaviridae core proteins remain favourite VLP candidates for the knowledge-based design of future vaccines, gene therapy vectors, specifically targeted nanocontainers, and other modern nanotechnological tools for prospective medical use.

HBV core particles as a carrier for B cell/T cell epitopes.

In the middle 80s, recombinant hepatitis B virus cores (HBc) gave onset to icosahedral virus-like particles (VLPs) as a basic class of non-infectious carriers of foreign immunological epitopes. The recombinant HBc particles were used to display immunodominant epitopes of hepatitis B, C, and E virus, human rhinovirus, papillomavirus, hantavirus, and influenza virus, human and simian immunodeficiency virus, bovine and feline leukemia virus, foot-and-mouth disease virus, murine cytomegalovirus and poliovirus, and other virus proteins, as well as of some bacterial and protozoan protein epitopes. Practical applicability of the HBc particles as carriers was enabled by their ability to high level synthesis and correct self-assembly in heterologous expression systems. The interest in the HBc VLPs was reinforced by the resolution of their fine structure by electron cryomicroscopy and X-ray crystallography, which revealed an unusual alpha-helical organization of dimeric units of HBc shells, alternative packing into icosahedrons with T = 3 and T = 4 symmetry, and the existence of long protruding spikes. The tips of the latter seem to be the optimal targets for the display of foreign sequences up to 238 amino acid residues in length. Combination of numerous experimental data on epitope display with the precise structural information enables a knowledge-based design of diagnostic, and vaccine and gene therapy tools on the basis of the HBc particles.

Expression, assembly competence and antigenic properties of hepatitis B virus core gene deletion variants from infected liver cells.

Previous studies have shown that the progression of hepatitis B virus-related liver disease in long-term immunosuppressed kidney transplant recipients is associated with the accumulation of virus variants carrying in-frame deletions in the central part of the core gene. A set of naturally occurring core protein variants was expressed in Escherichia coli in order to investigate their stability and assembly competence and to characterize their antigenic and immunogenic properties. In addition, a library of core gene variants generated in vitro with deletions including the major immunodominant region (MIR) of the core protein was investigated. The position and length of deletions determined the behaviour of mutant core proteins in E. coli and their assignment to one of the three groups: (i) assembly-competent, (ii) stable but assembly-incompetent and (iii) unstable proteins. In vivo core variants with MIR deletions between amino acids 77 and 93 belong to the first group. Only proteins with the shortest deletion (amino acids 86-93) showed stability and self-assembly at the same level as wild-type cores, and they showed reduced antigenicity and immunogenicity. Mutants with deletions extending N-terminally beyond residue G73 or C-terminally beyond G94 were found to be assembly-incompetent. We suggest that G73 and G94 are involved in the folding and the native assembly of core molecules, whereas the intervening sequence determines the antibody response. Depending on their ability to form stable proteins or to assemble into particles, core mutants could contribute to liver cell pathogenesis in different ways.

Behavior of a short preS1 epitope on the surface of hepatitis B core particles.

The major immunodominant region of hepatitis B core particles is widely recognized as the most prospective target for the insertion of foreign epitopes, ensuring their maximal antigenicity and immunogenicity. This region was mapped around amino acid residues 79-81, which were shown by electron cryo-microscopy to be located on the tips of the spikes protruding from the surface of hepatitis B core shells. Here we tried to expose a model sequence, the short immunodominant hepatitis B preS1 epitope 31-DPAFR-35, onto the tip of the spike, with simultaneous deletion of varying stretches from the major immunodominant region of the HBc molecule. Accessibility to the monoclonal anti-preS1 antibody MA18/7 and specific immunogenicity of the preS1 epitope depended on the location and length of the deletion. While chimeras with deletions within the stretch 79-88 presented the preS1 epitope on their surface and demonstrated remarkable preS1 immunogenicity, the corresponding chimeras without any deletion or with a more prolonged deletion (79-93) were unable to provide such presentation and possessed a lower specific preS1 immunogenicity. Deletion of the stretch 79-81 was sufficient to avoid the intrinsic HBc immunogenicity of the core particles, although chimeras with deleted major immunodominant region retained their property to be recognized by human polyclonal or hyperimmune anti-HBc antibodies.

Hepatitis B core particles as a universal display model: a structure-function basis for development.

Because it exhibits a remarkable capability to accept mutational intervention and undergo correct folding and self-assembly in all viable prokaryotic and eukaryotic expression systems, hepatitis B core (HBc) protein has been favored over other proposed particulate carriers. Structurally, the unusual alpha-helical organization of HBc dimeric units allows introduction of foreign peptide sequences into several areas of HBc shells, including their most protruding spikes. Progress toward full resolution of the spatial structure as well as accumulation of chimeric HBc-based structures has brought closer the knowledge-based design of future vaccines, gene therapy tools and other artificial particulate objects.

The natural 6 S RNA found in Q beta-infected cells is derived from host and phage RNA.

The RNA of Escherichia coli infected with RNA bacteriophage Q beta was isolated and screened for replicable short-chained RNA. In contrast to earlier assumptions we show that (i) short-chained replicable RNA is a very minor part of the RNA synthesized in the infection cycle, and (ii) that the replicable RNA isolated from infected cells is derived from cellular RNA, in particular 23 S rRNA and 10 Sa RNA, and from Q beta RNA itself. None of the many RNA species known from in vitro experiments was found. The RNA species isolated were all inefficient templates. No replicable RNA could be isolated from non-infected cells. Even in cells expressing high amounts of Q beta replicase very few RNA species could be isolated. RNA generated in vitro in template-free synthesis is therefore not derived from RNA species found in vivo, and replicable RNA found in vitro is generated by a mechanism fundamentally different from the one operating in vivo.

Spatial structure and insertion capacity of immunodominant region of hepatitis B core antigen.

Spatial and immunochemical elucidation of hepatitis B core antigen suggested unique organization of its major immunodominant region (MIR) localized within the central part of molecule around amino acid residues 74-83. This superficial loop was recognized as the most prospective target for the insertion of foreign epitopes ensuring maximal antigenicity and immunogenicity of the latter. MIR allowed a substantial capacity of insertions up to about 40 amino acid residues without loss of the capsid-forming ability of core particles. Vector capacity as well as structural behavior and immunological fate of inserted epitopes were dependent on their primary structure. Special sets of display vectors with retained but cross-sectioned MIR as well as with uni- and bidirectionally shortened MIR have been investigated.

Hepatitis B virus core particles as epitope carriers.

HBV core (HBc) particle is one of the most intensively studied particulate carriers for the insertion of foreign peptide sequences. Recombinant HBc protein expressed from the cloned gene undergoes the correct folding in a large variety of bacterial, yeast, insect and mammalian cells. Unique assembly properties and shape of 30/34-nm HBc particles allow substantial insertions into their primary structure without loss of their capsid-forming ability. N- and C-terminal regions, as well as the immunodominant loop in the middle of the molecule are widely accepted as targets for the introduction of foreign epitopes, ensuring retention and even enhancement of the original immunological activity of inserted sequences. Special sets of display vectors have been constructed on the basis of the cloned HBc gene. Epitope sequences of viral (BLV, FeLV, FMDV, HBV, HCV, HIV-1, HRV2, MCMV, PV-1, SIV) and nonviral (human chorionic gonadotropin) origin have been studied as model display moieties.

Structure and analysis of the 5' flanking region of the human interleukin-2 gene.

To investigate the structural and functional organization of the human interleukin-2 locus, the nucleotide sequence of 9339 bp of the 5' flanking region of this gene has been determined. Computer search analysis reveals five stretches with a high degree of homology between the human and mouse 5' flanking sequence, including a very distant 5' region. In this region additional binding sites for potential transcription factors were found that are identical to known regulatory sequences. The possible roles of these putative regulatory elements in the interleukin-2 gene regulation remain to be proven. The 5' end of the sequence contains almost full-length LINE element. LINE consensus open reading frames ORF1 and ORF2 in the reported sequence are interrupted by insertions, deletions or in-frame nonsense mutations. Comparative analysis of the ratio of codon changes that result in amino acid replacement to those that are silent revealed a high silent mutation frequency throughout the consensus ORF1 3' end, suggesting that this region is probably under selection for protein function.

Fine mapping and functional characterization of two immuno-dominant regions from the preS2 sequence of hepatitis B virus.

A set of monoclonal antibodies (mAbs) directed against the preS2 region of hepatitis B virus (HBV) surface antigen (HBsAg) was generated by immunization of mice with native HBsAg isolated from the blood of HBV carriers. According to (1) mutual competition binding of mAb to natural HBsAg, (2) recognition of full-length preS2 displayed on hepatitis B core particles, (3) recognition of synthetic partial preS2 peptides, and (4) Western blotting using a fusion protein library of truncated preS2 fragments of different legths, mAbs were assigned to two groups which coincided with groups I and III described by Mimms et al. [Virology 1990; 176:604-619]. All mAbs recognized linear epitopes and were glycosylation independent. Six out of eight fine-mapped mAbs recognized common epitopes located in the amino-terminal part of the preS sequence between amino acids 131 and 144 (group I), and inhibited binding of HBsAg to polymerized human serum albumin. Only two mAbs recognized a carboxy-terminal HBV-genotype-specific epitope covering amino acid residues 162 to 168 (group III). These mAbs bound to the highly variable proteolysis-sensitive hinge of preS2. Although four out of six mAbs targeted to immunodominant region I require the full-length sequence 131-L[Q/L]DPRVRGLY[F/L]PAG-144, two mAbs recognize the shorter and slightly carboxy-terminal-shifted sequences 133-DPRVRGLY[F/L]-141 or 135-PVRGLY[F/L]PAG-144. Together with previously identified preS2 epitopes 133-DPRVRGL-139, 137-RGLYFPA-143, and 132-QDPR-135, these data indicate diversity of the immune response against epitopes within the same immunodominant region. This diversity may be generated by a labile secondary structure. Sequence analysis suggests the transition from an alpha-helix to a loop structure at this site.

Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis.

A structure-function analysis of the icosahedral RNA bacteriophage fr coat protein (CP) assembly was undertaken using linker-insertion, deletion and substitution mutagenesis. Mutations were specifically introduced into either pre-existing or artificially created restriction enzyme sites within fr CP gene expressed in Escherichia coli from a recombinant plasmid. This directs synthesis of wild type protein that undergoes self-assembly and forms capsid-like particles indistinguishable morphologically and immunologically from native phage particles. A series of fr CP variants containing sequence alterations in the regions which are (i) exposed on the external surface of capsid or (ii) located on the contacting areas between CP subunits were obtained and their assembly properties investigated. The majority of mutants demonstrated reduction of assembly ability and formed either CP dimers (mutations at residues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69). The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of the fr CP. A third type of assembled structure was formed by a variant with a single amino acid substitution I104T. The alpha A-helix region (residues 97-111) is particularly sensitive to mutation and any alteration in this region decreases accumulation of mutant protein in E. coli. The relative contributions of particular fr CP domains in maintenance of capsid structural integrity as well as the possible capsid assembly mechanism are discussed.

Immunochemical structure of the carboxy-terminal part of hepatitis B e antigen: identification of internal and surface-exposed sequences.

The C-terminal region of hepatitis B virus (HBV) e antigen (HBeAg), amino acids (aa) 121 to 147, was characterized for reactivity with 15 monoclonal antibodies (MAbs) and sera from 16 chronic carriers on the HB surface antigen with anti-HBe. Recombinant proteins exposing fragments of the HBc/e polypeptide (aa 29 to 176, 60 to 176, 101 to 176, 121 to 176, 134 to 176, 138 to 176, 139 to 176, 140 to 176, 146 to 176 and 156 to 176) fused to the N terminus of the coat protein of RNA phage fr were constructed, as were two sets of synthetic peptides covering residues 121 to 136 and 130 to 147, where each residue was sequentially substituted by alanine. The MAbs were found to recognize overlapping epitopes in the fusion proteins within residues 121 to 176; however, none of the MAbs reacted with proteins covering residues 146 to 176 and 156 to 176. Using the synthetic peptides it was found that the MAbs recognized epitopes at residues 128-TPPAYR-133, 133-RPPNAP-138, 135-PNAPIL-140, 138-PILSTLPE-145 and 143-LPET-146. Only MAbs recognizing the epitope 128-TPPAYR-133 were found to react with both native HBeAg and denatured HBcAG, whereas MAbs recognizing epitopes located closer to the C terminus of HBeAg were reactive only with denatured HBcAg. The recognition sites for the human IgG1 overlapped with the epitopes of the MAbs recognizing native HBeAg. Our interpretation of these findings is that the region 124 to 133 is on the surface of native HBeAg and denatured HBcAg, and that the adjacent region 135 to 147 is not accessible on the surface of native HBeAg, but becomes exposed on denatured HBcAg.

Hybrid hepatitis B virus nucleocapsid bearing an immunodominant region from hepatitis B virus surface antigen.

A hepatitis B core antigen (HBcAg) gene bearing the 39-amino-acid-long domain A of hepatitis B surface antigen (HBsAg) within the HBcAg immunodominant loop has been constructed and expressed in Escherichia coli. Chimeric capsids demonstrated HBs but not HBc antigenicity and elicited in mice B-cell and T-cell responses against native HBcAg and HBsAg.