Molecular basis of the interaction of the human tyrosine phosphatase PTPN3 with the hepatitis B virus core protein.

Interactions between the hepatitis B virus core protein (HBc) and host cell proteins are poorly understood, although they may be essential for the propagation of the virus and its pathogenicity. HBc has a C-terminal PDZ (PSD-95, Dlg1, ZO-1)-binding motif (PBM) that is responsible for interactions with host PDZ domain-containing proteins. In this work, we focused on the human protein tyrosine phosphatase non-receptor type 3 (PTPN3) and its interaction with HBc. We solved the crystal structure of the PDZ domain of PTPN3 in complex with the PBM of HBc, revealing a network of interactions specific to class I PDZ domains despite the presence of a C-terminal cysteine in this atypical PBM. We further showed that PTPN3 binds the HBc protein within capsids or as a homodimer. We demonstrate that overexpression of PTPN3 significantly affects HBV infection in HepG2 NTCP cells. Finally, we performed proteomics studies on both sides by pull-down assays and screening of a human PDZ domain library. We identified a pool of human PBM-containing proteins that might interact with PTPN3 in cells and that could be in competition with the HBc PBM during infection, and we also identified potential cellular partners of HBc through PDZ-PBM interactions. This study opens up many avenues of future investigations into the pathophysiology of HBV.

BAY 41-4109-mediated aggregation of assembled and misassembled HBV capsids in cells revealed by electron microscopy.

HBc is a small protein essential for the formation of the icosahedral HBV capsid. Its multiple roles in the replication cycle make this protein a promising target for the development of antiviral molecules. Based on the structure of HBc, a series of HBV assembly inhibitors, also known as capsid assembly modulators, were identified. We investigated the effect of BAY 41-4109, a heteroaryldihydropyrimidine derivative that promotes the assembly of a non-capsid polymer. We showed, by confocal microscopy, that BAY 41-4109 mediated HBc aggregation, mostly in the cytoplasm of Huh7 cells. Image analysis revealed that aggregate size depended on BAY 41-4109 concentration and treatment duration. Large aggregates in the vicinity of the nucleus were enclosed by invaginations of the nuclear envelope. This deformation of the nuclear envelope was confirmed by transmission electron microscopy (TEM) and immuno-TEM. These two techniques also revealed that the HBc aggregates were accumulations of capsid-like shells with an electron-dense material consisting of HBV core fragments. These findings, shedding light on the ultrastructural organization of HBc aggregates, provide insight into the mechanisms of action of BAY 41-4109 against HBV and will serve as a basis for comparison with other HBV capsid assembly inhibitors.

The Structural Biology of Hepatitis B Virus: Form and Function.

Hepatitis B virus is one of the smallest human pathogens, encoded by a 3,200-bp genome with only four open reading frames. Yet the virus shows a remarkable diversity in structural features, often with the same proteins adopting several conformations. In part, this is the parsimony of viruses, where a minimal number of proteins perform a wide variety of functions. However, a more important theme is that weak interactions between components as well as components with multiple conformations that have similar stabilities lead to a highly dynamic system. In hepatitis B virus, this is manifested as a virion where the envelope proteins have multiple structures, the envelope-capsid interaction is irregular, and the capsid is a dynamic compartment that actively participates in metabolism of the encapsidated genome and carries regulated signals for intracellular trafficking.

A VLP library of C-terminally truncated Hepatitis B core proteins: correlation of RNA encapsidation with a Th1/Th2 switch in the immune responses of mice.

An efficient pBR327- and Ptrp-based E. coli expression system was used to generate a large-scale library of virus like particles (VLP) formed by recombinant hepatitis B virus (HBV) core (HBc) protein derivatives. To construct the library, the gene of HBc protein of the genotype D/subtype ayw2 virus was gradually truncated from the 3`-end and twenty-two HBc variants (with truncation up to 139 aa) were expressed at high levels. The proteins were purified by salt precipitation and gel filtration. Background RNA binding was observed for VLPs formed by HBc1-149, which lacked all C-terminal Arg blocks, and the addition of three Arg residues (HBc1-152) only slightly increased RNA binding. The presence of two Arg blocks (proteins HBc1-162 and HBc1-163) resulted in approximately half of the typical level of RNA binding, and the presence of three blocks (protein HBc1-171) led to approximately 85% of the typical level of binding. Only a small increase in the level of RNA binding was found for the HBc1-175 VLPs, which contained all four Arg blocks but lacked the last 8 aa of the full-length HBc protein. VLPs containing high levels of RNA had higher antigenicity according to an ELISA with anti-HBc mAbs than the VLPs formed by HBc variants without C-terminal Arg blocks and lacking RNA. The results indicate that the VLPs were stabilised by nucleic acids. The immunogenicity in BALB/c mice was comparable for VLPs formed by different HBc proteins, but a clear switch from a Th1 response to a Th2 response occurred after the loss of encapsidated RNA. We did not observe significant differences in lymphocyte proliferation in vitro for the tested VLP variants; however, the loss of RNA encapsidation correlated with a decreased level of IFN-γ induction, which is a measure of the potential CTL activity of immunogens.

Three-dimensional structure of the hepatitis B core antigen particle truncated at residue 154.

The three-dimensional structure of recombinant hepatitis B core antigen (HBcAg) particles truncated at residue 154 (HBcAg-154) was determined to 7.8 Å resolution by cryo-electron microscopy (cryoEM) and computer reconstruction. The capsid of HBcAg-154 is mainly constituted by α-helical folds, highly similar to that of HBcAg-149. The C-terminal region between residues 155 and 183 of the core protein is more crucial to the encapsidation of RNA, and the short C-terminal tail of HBcAg-154 results in a nearly empty capsid.

Structural comparisons of hepatitis B core antigen particles with different C-terminal lengths.

Core protein of hepatitis B virus (HBV) with various C-terminal lengths (residue 154, 164, 167 and 183) can self-assemble into recombinant hepatitis B core antigen (HBcAg) particles. To understand the RNA encapsidation mechanism of HBV, the three-dimensional structures of these particles were reconstructed by cryo-electron microscopy (cryoEM). Detailed structural comparisons showed that their capsid structures are highly similar, while the RNA content is increased upon the retention of more amino acid residues at the C-terminus of core protein, suggesting the crucial role of the basic C-terminal tail on determining the genome size.

Enhanced capacity of antigen presentation of HBc-VLP-pulsed RAW264.7 cells revealed by proteomics analysis.

Many recent studies have indicated that virus-like particles (VLPs) have many potential applications in the fields of vaccine development and gene therapy. However, we still know little about the subtle mechanisms involved in the presentation of VLPs by antigen presenting cells (APCs). To illustrate the mechanisms, we utilized two-dimensional electrophoresis and tandem MS to compare and identify differentially expressed proteins between hepatitis B virus core antigen VLP (HBc-VLP)-pulsed and control RAW264.7 cells. Of the 25 spots identified as differentially expressed ( p < 0.05) between the two cell lines, 11 (corresponding to 11 unique proteins) were positively identified. Further analysis of two proteins, prohibitin and heat shock protein 70, confirmed that these proteins are expressed at higher levels in HBc-VLP-pulsed RAW264.7 cells compared with control cells. The proteins identified in this study will be useful in revealing the mechanisms that underlie VLP-APC interactions. Overall, this study also provides some useful suggestions for vaccine development and gene therapy.

Recombinant hepatitis B virus core particles: association, dissociation and encapsidation of green fluorescent protein.

The recombinant hepatitis B virus (HBV) core antigen (HBcAg) expressed in Escherichia coli self-assembles into icosahedral capsids of about 35 nm which can be exploited as gene or drug delivery vehicles. The association and dissociation properties of the C-terminally truncated HBcAg with urea and guanidine hydrochloride (GdnHCl) were studied. Transmission electron microscopy (TEM) revealed that the dissociated HBcAg was able to re-associate into particles when the applied denaturing agents were physically removed. In order to evaluate the potential of the particles in capturing molecules, purified green fluorescent protein (GFP) was applied to the dissociated HBcAg for encapsidation. The HBcAg particles harbouring the GFP molecules were purified using sucrose density gradient ultracentrifugation and analysed using native agarose gel electrophoresis and TEM. A method for the encapsidation of GFP in HBcAg particles which has the potential to capture drugs or nucleic acids was established.

Expression and purification of the recombinant HBcAg core particles derived from methyltrophic Pichia pastoris, and TEM and AFM of the core particles and their natural aggregates.

The recombinant hepatitis B virus core antigen (rHBcAg) core particles derived from Pichia pastoris were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicate that the purified rHBcAg particles with HBcAg antigenicity mainly locate at the densities of 1.2576 and 1.3013 g.mL(-1), respectively. After purification, a portion of purified sample of rHBcAg particles was immediately subjected to detection using transmission electron microscopy (TEM) and atomic force microscopy (AFM), the remainder were kept in -20 degrees C for 1 month or longer. After 30 days, the sample of rHBcAg particles previously frozen was imaged by TEM and AFM. The detection results indicate that the stored rHBcAg particles aggregated into a string of beads. The above results suggested that the rHBcAg particles expressed and self-assembled in P. pastoris, which were stored at -20 degrees C, can gradually and naturally aggregate with storage time.

Epitope diversity of hepatitis B virus capsids: quasi-equivalent variations in spike epitopes and binding of different antibodies to the same epitope.

To investigate the range of antigenic variation of HBV capsids, we have characterized the epitopes for two anti-capsid antibodies by cryo-electron microscopy and image reconstruction of Fab-labeled capsids to approximately 10A resolution followed by molecular modeling. Both antibodies engage residues on the protruding spikes but their epitopes and binding orientations differ. Steric interference effects limit maximum binding to approximately 50% average occupancy in each case. However, the occupancies of the two copies of a given epitope that are present on a single spike differ, reflecting subtle distinctions in structure and hence, binding affinity, arising from quasi-equivalence. The epitope for mAb88 is conformational but continuous, consisting of a loop-helix motif (residues 77-87) on one of the two polypeptide chains in the spike. In contrast, the epitope for mAb842, like most conformational epitopes, is discontinuous, consisting of a loop on one polypeptide chain (residues 74-78) combined with a loop-helix element (residues 78-83) on the other. The epitope of mAb842 is essentially identical with that previously mapped for mAb F11A4, although the binding orientations of the two monoclonal antibodies (mAbs) differ, as do their affinities measured by surface plasmon resonance. From the number of monoclonals (six) whose binding had to be characterized to give the first duplicate epitope, we estimate the total number of core antigen (cAg) epitopes to be of the order of 20. Given that different antibodies may share the same epitope, the potential number of distinct anti-cAg clones should be considerably higher. The observation that the large majority of cAg epitopes are conformational reflects the relative dimensions of a Fab (large) and the small size and close packing of the motifs that are exposed and accessible on the capsid surface.

Purification of the recombinant hepatitis B virus core antigen (rHBcAg) produced in the yeast Saccharomyces cerevisiae and comparative observation of its particles by transmission electron microscopy (TEM) and atomic force microscopy (AFM).

Hepatitis B virus core antigen (HBcAg) gene (C gene) was expressed in Saccharomyces cerevisiae and the products (rHBcAg or core particles) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, Sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. It has been observed that HBcAg was synthesized in yeast cells as a particle consisting of polypeptides with a molecular weight of 21.5 kDa (p21.5). Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (rHBcAg particles) with HBcAg antigenicity mainly located at the densities of 1.27 and 1.40 g ml(-1), respectively. Observation and analysis of the purified rHBcAg products by TEM indicated that rHBcAg peptides could mainly self-assemble into two size classes of core particles. The larger particles were approximately 30.1 nm and the smaller were approximately 21.5 nm in mean diameter. Further observation and analysis of the same rHBcAg (core) particles by AFM also indicated that rHBcAg (core) particles were similar to the native HBcAg (core) particles from infected human hepatocytes and mainly composed of two size classes of partides core. The larger particles were approximately 31.3 nm and the smaller were approximately 22.5 nm in mean diameter which was similar to the results obtained by TEM. All results from both TEM and AFM suggested that core particles (capsids) produced in S. cerevisiae possessed dimorphism.

Stability and morphology comparisons of self-assembled virus-like particles from wild-type and mutant human hepatitis B virus capsid proteins.

Instead of displaying the wild-type selective export of virions containing mature genomes, human hepatitis B virus (HBV) mutant I97L, changing from an isoleucine to a leucine at amino acid 97 of HBV core antigen (HBcAg), lost the high stringency of selectivity in genome maturity during virion export. To understand the structural basis of this so-called "immature secretion" phenomenon, we compared the stability and morphology of self-assembled capsid particles from the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183) or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using negative staining and electron microscopy, full-length particles appear as "thick-walled" spherical particles with little interior space, whereas truncated particles appear as "thin-walled" spherical particles with a much larger inner space. We found no significant differences in capsid stability between wild-type and mutant I97L particles under denaturing pH and temperature in either full-length or truncated core protein contexts. In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at temperatures higher than 75 degrees C, or in 0.1% sodium dodecyl sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated full-length particles during agarose gel electrophoresis is most likely caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids are known to exist in natural infection as dimorphic T=3 or T=4 icosahedral particles. No difference in the ratio between T=3 (78%) and T=4 particles (20.3%) are found between wild-type HBV and mutant I97L in the context of HBcAg1-140. In addition, we found no difference in capsid stability between T=3 and T=4 particles successfully separated by using a novel agarose gel electrophoresis procedure.

The morphogenic linker peptide of HBV capsid protein forms a mobile array on the interior surface.

Many capsid proteins have peptides that influence their assembly. In hepatitis B virus capsid protein, the peptide STLPETTVV, linking the shell-forming 'core' domain and the nucleic acid-binding 'protamine' domain, has such a role. We have studied its morphogenic properties by permuting its sequence, substituting it with an extraneous peptide, deleting it to directly fuse the core and protamine domains and assembling core domain dimers with added linker peptides. The peptide was found to be necessary for the assembly of protamine domain-containing capsids, although its size-determining effect tolerates some modifications. Although largely invisible in a capsid crystal structure, we could visualize linker peptides by cryo-EM difference imaging: they emerge on the inner surface and extend from the capsid protein dimer interface towards the adjacent symmetry axis. A closely sequence-similar peptide in cellobiose dehydrogenase, which has an extended conformation, offers a plausible prototype. We propose that linker peptides are attached to the capsid inner surface as hinged struts, forming a mobile array, an arrangement with implications for morphogenesis and the management of encapsidated nucleic acid.

Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles.

The simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co-expression of wt and a mutant core lacking 17 amino acid residues (77-93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly.

Purification of E. coli-expressed HIS-tagged hepatitis B core antigen by Ni2+ -chelate affinity chromatography.

Hepatitis B virus is a major cause of human liver disease. In the case of chronic infection the virus can lead to liver cancer and cirrhosis. The virion consists of an outer envelope containing lipids of the endoplasmic reticulum and virally-encoded surface proteins. This lipoprotein shell encloses the nucleocapsid or core antigen (HBcAg), which contains the viral genome. The capsid consists of dimers of a 183-residue protein, which can be divided into an assembly (residues 1-149) and a protamin-like domain (residues 150-183), responsible for polymerization into particles and RNA packaging, respectively. Upon expression of the core gene in bacteria the products are assembled into capsids resembling those of wild type particles. A purification protocol was developed for unpolymerised (dimeric) and polymerized HBcAg by fusion of six histidine residues to a C-terminal deletion mutant of the core protein allowing the isolation of the respective antigens after denaturing Ni2+-chelate affinity chromatography and renaturing dialysis. The possible incorporation of E. coli proteins during the assembly process and the inclusion of nucleic acids can be avoided. The method might be an attractive alternative to common purification protocols of hybrid virus-like particles (VLPs) for vaccine use.

Native display of complete foreign protein domains on the surface of hepatitis B virus capsids.

The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein's primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.

Specificity of humoral and cellular immune response against recombinant particles of nucleocapsid protein of human hepatitis B virus in rabbits.

Nucleocapsid (core) protein of hepatitis B virus (HBcAg) induces potent cellular and humoral responses that have a clear protective potential. Rabbits were immunized by particles formed by recombinant molecules of HBcAg carrying N-terminally inserted heterologous sequences. Specificity of humoral and cellular immune response against HBcAg and selection of HBcAg epitopes was surveyed. Immunological properties of the recombinant particles were similar to those of the original HBcAg. Recombinant particles were not toxic to the peripheral blood mononuclear cells (PBMC) of non-immune or HBcAg-immunized animals ex vivo. Proliferative response of PBMC (T-lymphocytes) to HBcAg in immunized animals increased in a concentration-dependent manner in the broad interval of HBcAg concentrations (10-104 ng/ml). On the contrary, a narrow bell-shaped HBcAg dose-dependence curve was earlier observed for T-lymphocytes of donors immune to HBV after natural infection that was probably due to the cytotoxic effect of HBcAg on the expressing cells. Specificity of humoral and cellular immune response against HBcAg particles in the immunized animals and in natural infection with hepatitis B virus (HBV) was compared. Immunization with recombinant HBcAg particles induced potent anti-HBcAg antibody responses: high (up to 2.107) titers of anti-HBcAg antibodies were reached. Appearance of anti-HBcAg antibodies was in every case preceded by an increasing T-cell response to the whole protein and HBcAg-derived peptides, thus mimicking immune responses during acute HBV infection in humans. A predominant universal (haplotype-independent) T-helper cell epitope (amino acid residues (aa) 61-85 of HBcAg (p61-85)) was recognized by T-cells of all animals. Transient antibody response against p61-85 was recorded during the early stages of immunization in spite of the fact that a major B-cell epitope localized in this region is supposed to be purely conformational. A sequence representing another cluster of immunodominant T-cell epitopes of mice and HBV infected humans, aa 121-140 (p121-140), was not immunogenic on the T-cell level. However, it appeared to be a potent B-cell immunogen, despite a common assumption that HBcAg and p121-140 are not cross-reactive at the B-cell level. A possibility that anti-p121-140 antibodies were induced by an exposed region of the native particulate HBcAg and not by the denatured protein molecules, was confirmed by recognition of the particulate HBcAg by antibodies specific to synthetic peptides representing aa 120-140 of HBcAg. The data point to the exposition of aa 121-140 on the surface of the particles.

Mosaic hepatitis B virus core particles allow insertion of extended foreign protein segments.

Because of its particular immunological properties, the core protein of hepatitis B virus (HBcAg) has become one of the favoured 'virus-like particles' for use as a carrier of foreign epitopes. A new strategy to construct core particles presenting extended foreign protein segments was established based on the introduction of a linker containing a translational stop codon between sequences encoding a C-terminally truncated HBcAg (HBcAg delta) and a foreign protein sequence. Expression in an Escherichia coli suppressor strain allowed the simultaneous synthesis of both HBcAg delta and a read-through fusion protein containing a part of the hantavirus nucleocapsid protein. After purification, the presence of core-like mosaic particles with HBc and hantavirus antigenicity was demonstrated by electron microscopy and immunological tests. This strategy of partial stop codon suppression should improve the use of HBcAg as a carrier of foreign epitopes by allowing insertion of long foreign sequences into particle-forming proteins. The resulting mosaic particles should be of general interest for further vaccine developments.

Simple method for efficient production of hepatitis B virus core antigen in Escherichia coli.

To obtain good antigenicity and high purity of the hepatitis B virus core antigen (HBcAg) in large quantities without using the fused protein technique employed in recombinant DNA technology, a protein molecule with the same primary sequence as that of wild-type HBcAg (subtype adr) was directly expressed in Escherichia coli JM109 (DE3) using pGd1 expression vector. Purification of the expressed HBcAg yielded high-quality protein by means of simple purification steps, such as sonication, ammonium sulphate precipitation and heat treatment, before final purification by conventional ultra-centrifugation. The HBcAg preparation thus obtained contains small round particles similar in appearance to the HBcAg particles from the HBV-infected human liver tissue.