Hepatitis B Virus Core Particles Research Articles

Anti-HBV Activities of Cembranoids from the South China Sea Soft Coral Sinularia pedunculata and Their Structure Activity Relationship.

Hepatitis B Virus (HBV) infection is a global public health challenge that seriously endangers human health. Soft coral, as a major source of terpenoids, contains many structurally novel and highly bioactive compounds. Sixteen cembranoids (1-16), including a new one named sinupedunol B (16), were isolated from the South China Sea Soft coral Sinularia pedunculata. The structure of the sinupedunol B (16) was determined through a combination of spectroscopic analysis and X-ray single-crystal diffraction. In this study, cembranoids isolated from Sinularia pedunculata were found of anti-HBV activity for the first time. Among them, flexilarin D (6) showed significant anti-HBV activity with an IC50 value of 5.57 μM without cytotoxicity. We then analyzed the structure-activity relationship (SAR). Furthermore, it is demonstrated that flexilarin D (6) can accelerate the formation of capsid, inhibit HBeAg, HBV core particle DNA, HBV total RNA and pregenomic RNA in a dose dependent manner. We also confirmed the anti-HBV activity of 6 in HepG2-NTCP infection system. Finally, we demonstrated the anti-HBV mechanism of these compounds by inhibiting the ENI/Xp enhancer/promoter.

Development and characterization of monoclonal antibody against the critical loop structure of african swine fever virus P72 protein

African swine fever (ASF) is a highly infectious and lethal viral disease caused by the African swine fever virus (ASFV). The four prominent loop structures on the surface of the primary structural protein P72 are considered to be key protective epitopes. In this study, the four critical loops (ER1–4) of the ASFV p72 protein were individually fused to hepatitis B virus core particles (HBc) and self-assembled into nanoparticles to preserve the natural conformation of the loop structure and enhance its immunogenicity. Then, four recombinant proteins were obtained in E. coli expression system and monoclonal antibodies (mAbs) were developed and characterized. All 10 mAbs obtained were able to react with P72 protein and ASFV with potencies up to 1:204 800. Amino acids 250–274, 279–299 and 507–517 of the P72 protein were identified as linear epitopes and highly conserved. The mAb 4G8 showed the highest inhibition rate of 84% against ASFV positive sera. Importantly, neutralization experiments illustrated that mAb 4G8 has a 67% inhibition rate, indicating that its corresponding epitopes are potential candidates for ASFV vaccine. In conclusion, highly immunogenic nanoparticles of the ASFV P72 key loop were constructed to induce the production of highly effective mAbs and clarify their epitope information for the diagnosis and prevention of ASFV.

Efficient stabilization of therapeutic hepatitis B vaccine components by amino-acid formulation maintains its potential to break immune tolerance

Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach. We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice. Invitro analysis proved the vaccine's stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40°C for 3 months and at 25°C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4°C/-80°C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice. SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide. Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world.

Autophagic membranes participate in hepatitis B virus nucleocapsid assembly, precore and core protein trafficking, and viral release

Hepatitis B virus (HBV) DNA replication takes place inside the viral core particle and is dependent on autophagy. Here we show that HBV core particles are associated with autophagosomes and phagophores in cells that productively replicate HBV. These autophagic membrane-associated core particles contain almost entirely the hypophosphorylated core protein and are DNA replication competent. As the hyperphosphorylated core protein can be localized to phagophores and the dephosphorylation of the core protein is associated with the packaging of viral pregenomic RNA (pgRNA), these results are in support of the model that phagophores can serve as the sites for the packaging of pgRNA. In contrast, in cells that replicate HBV, the precore protein derivatives, which are related to the core protein, are associated with autophagosomes but not with phagophores via a pathway that is independent of its signal peptide. Interestingly, when the core protein is expressed by itself, it is associated with phagophores but not with autophagosomes. These observations indicate that autophagic membranes are differentially involved in the trafficking of precore and core proteins. HBV induces the fusion of autophagosomes and multivesicular bodies and the silencing of Rab11, a regulator of this fusion, is associated with the reduction of release of mature HBV particles. Our studies thus indicate that autophagic membranes participate in the assembly of HBV nucleocapsids, the trafficking of HBV precore and core proteins, and likely also the egress of HBV particles.

Hepatitis B virus virion secretion is a CRM1-spike-mediated late event

BackgroundHepatitis B virus (HBV) is a major human pathogen worldwide. To date, there is no curative treatment for chronic hepatitis B. The mechanism of virion secretion remains to be investigated. Previously, we found that nuclear export of HBc particles can be facilitated via two CRM1-specific nuclear export signals (NES) at the spike tip.MethodsIn this study, we used site-directed mutagenesis at the CRM1 NES, as well as treatment with CRM1 inhibitors at a low concentration, or CRM1-specific shRNA knockdown, in HBV-producing cell culture, and measured the secretion of various HBV viral and subviral particles via a native agarose gel electrophoresis assay. Separated HBV particles were characterized by Western blot analysis, and their genomic DNA contents were measured by Southern blot analysis. Secreted extracellular particles were compared with intracellular HBc capsids for DNA synthesis and capsid formation. Virion secretion and the in vivo interactions among HBc capsids, CRM1 and microtubules, were examined by proximity ligation assay, immunofluorescence microscopy, and nocodazole treatment.ResultsWe report here that the tip of spike of HBV core (HBc) particles (capsids) contains a complex sensor for secretion of both HBV virions and naked capsids. HBV virion secretion is closely associated with HBc nuclear export in a CRM1-dependent manner. At the conformationally flexible spike tips of HBc particles, NES motifs overlap extensively with motifs important for secretion of HBV virions and naked capsids.ConclusionsWe provided experimental evidence that virions and naked capsids can egress via two distinct, yet overlapping, pathways. Unlike the secretion of naked capsids, HBV virion secretion is highly CRM1- and microtubule-dependent. CRM1 is well known for its involvement in nuclear transport in literature. To our knowledge, this is the first report that CRM1 is required for virion secretion. CRM1 inhibitors could be a promising therapeutic candidate for chronic HBV patients in clinical medicine.

Long-term and efficient inhibition of hepatitis B virus replication by AAV8-delivered artificial microRNAs

Chronic hepatitis B virus (HBV) infection remains a global health problem and current treatments are insufficient due to immune tolerance to hepatitis B surface antigen (HBsAg). RNA interference (RNAi) is a more promising approach for antiviral therapy. Here, 17 single artificial microRNAs (amiRNAs) targeting the highly conserved regions of HBV genome were screened to inhibit HBV replication. In addition, we compared three tandem amiRNAs, each containing 3 different amiRNAs, out of which amiRNA135 was selected to be studied in detail. In vitro data showed that amiRNA135 significantly inhibited the replication of different HBV genotypes (including resistant and mutant). In vivo study was carried out by adeno-associated virus 8-mediated gene delivery, we found that the anti-HBV effects of AAV8-amiRNA135 were time and dose-dependent. Serum HBsAg and HBeAg in high dose groups were significantly reduced at 7 days after a single intravenous vector injection, and maintained at low levels throughout a 15-month experiment. Immunohistochemical staining and HBV core particle DNA analysis confirmed that HBV replication in the liver was strongly inhibited by AAV8-amiRNA135. Taken together, our data suggest that AAV8-mediated trimeric amiRNA expression is a promising therapeutic approach for chronic HBV infection.

CRM1-spike-mediated nuclear export of hepatitis B virus encapsidated viral RNA.

Hepatitis B virus (HBV) is a global pathogen. We report here that the cellular CRM1 machinery can mediate nuclear export of entire HBV core (HBc) particles containing encapsidated viral RNAs. Two CRM1-mediated nuclear export signals (NESCRM1) cluster at the conformationally flexible spike tips of HBc particles. Mutant NESCRM1 capsids exhibit strongly reduced associations with CRM1 and nucleoporin358 invivo. CRM1 and NXF1 machineries mediate nuclear export of HBc particles independently. Inhibition of nuclear export has pleiotropic consequences, including nuclear accumulation of HBc particles, a significant reduction of encapsidated viral RNAs in the cytoplasm but not in the nucleus, and barely detectable viral DNA. We hypothesize an HBV life cycle where encapsidation of the RNA pregenome can initiate early in the nucleus, whereas DNA genome maturation occurs mainly in the cytoplasm. We identified a druggable target for HBV by blocking its intracellular trafficking.

Bioengineering of virus-like particles as dynamic nanocarriers for in vivo delivery and targeting to solid tumours.

Virus-like particles (VLPs) are known as self-assembled, non-replicative and non-infectious protein particles, which imitate the formation and structure of original wild type viruses, however, lack the viral genome and/or their fragments. The capacity of VLPs to encompass small molecules like nucleic acids and others has made them as novel vessels of nanocarriers for drug delivery applications. In addition, VLPs surface have the capacity to achieve variation of the surface display via several modification strategies including genetic modification, chemical modification, and non-covalent modification. Among the VLPs nanocarriers, Hepatitis B virus core (HBc) particles have been the most encouraging candidate. HBc particles are hollow nanoparticles in the range of 30-34nm in diameter and 7nm thick envelopes, consisting of 180 or 240 copies of identical polypeptide monomer. They also employ a distinctive position among the VLPs carriers due to the high-level synthesis, which serves as a strong protective capsid shell and efficient self-assembly properties. This review highlights on the bioengineering of HBc particles as dynamic nanocarriers for in vivo delivery and specific targeting to solid tumours.

Analysis of the Physicochemical Properties, Replication and Pathophysiology of a Massively Glycosylated Hepatitis B Virus HBsAg Escape Mutant.

Mutations in HBsAg, the surface antigen of the hepatitis B virus (HBV), might affect the serum HBV DNA level of HBV-infected patients, since the reverse transcriptase (RT) domain of HBV polymerase overlaps with the HBsAg-coding region. We previously identified a diagnostic escape mutant (W3S) HBV that produces massively glycosylated HBsAg. In this study, we constructed an HBV-producing vector that expresses W3S HBs (pHB-W3S) along with a wild-type HBV-producing plasmid (pHB-WT) in order to analyze the physicochemical properties, replication, and antiviral drug response of the mutant. Transfection of either pHB-WT or W3S into HepG2 cells yielded similar CsCl density profiles and eAg expression, as did transfection of a glycosylation defective mutant, pHB-W3S (N146G), in which a glycosylation site at the 146aa asparagine (N) site of HBs was mutated to glycine (G). Virion secretion, however, seemed to be severely impaired in cases of pHB-W3S and pHB-W3S (N146G), compared with pHB-WT, as determined by qPCR and Southern blot analysis. Furthermore, inhibition of glycosylation using tunicamycinTM on wild-type HBV production also reduced the virion secretion. These results suggested that the HBV core and Dane particle could be formed either by massively glycosylated or glycosylation-defective HBsAg, but reduced and/or almost completely blocked the virion secretion efficiency, indicating that balanced glycosylation of HBsAg is required for efficient release of HBV, and mutations inducing an imbalanced glycosylation of HBs would cause the virion to become stuck in the cells, which might be associated with various pathogeneses due to HBV infection.

A tumor-specific neoepitope expressed in homologous/self or heterologous/viral antigens induced comparable effector CD8+ T-cell responses by DNA vaccination

Somatic mutations in tumors often generate neoproteins that contain MHC-I-binding neoepitopes. Little is known if and how efficient tumor-specific neoantigens activate CD8+ T cells. Here, we asked whether a de novo generated neoepitope, encoded either within an otherwise conserved and ubiquitously expressed self-antigen or in a chimeric HBV core antigen expression platform, providing heterologous helper functions, induces CD8+ T cells in C57Bl/6J mice by DNA immunization. For it, we chose an established Db/Sp244-252/R251H neoepitope generated in the murine Endophilin-B2/SH3GLB2 (EndoB2-Sp) protein by a single amino acid exchange. We showed that a single injection of EndoB2-Sp expression vectors efficiently primed dimer/pentamer+, IFN-γ+ and cytolytic Db/Sp244-252/R251H-specific effector CD8+ T cells in C57Bl/6J mice. Priming of Db/Sp244-252/R251H-specific CD8+ T cells proceeded independent from CD4+ T-cell help in MHC-II-deficient Aα-/- mice. As compared to the homologous EndoB2-Sp vaccine, the selective expression of the Db/Sp244-252/R251H neoepitope in chimeric particle-forming and assembly-deficient HBV core antigens induced comparable frequencies Db/Sp244-252/R251H-specific CD8+ T cells with the same cytolytic effector phenotype. The homologous EndoB2 carrier, but not the nine-residue neoepitope presented on chimeric HBV core particles, induced EndoB2-specific IgG antibody responses. The HBV core expression platform is thus an attractive option to selectively induce neoepitope-specific effector CD8+ T cells by DNA vaccination. These novel findings have practical implications for the design of heterologous/self and heterologous/viral cancer vaccines that prime and/or activate neoepitope-specific CD8+ T cells.

Engineering Human Epidermal Growth Receptor 2-Targeting Hepatitis B Virus Core Nanoparticles for siRNA Delivery in Vitro and in Vivo.

Hepatitis B virus core (HBc) particles acquire the capacity to disassemble and reassemble in a controlled manner, allowing entrapment and delivery of drugs and macromolecules to cells. HBc particles are made of 180–240 copies of 21 kDa protein monomers, assembled into 30–34 nm diameter icosahedral particles. In this study, we aimed at formulating HBc particles for the delivery of siRNA for gene silencing in vitro and in vivo. We have previously reported recombinant HBc particles expressing ZHER2 affibodies, specifically targeting human epidermal growth receptor 2 (HER2)-expressing cancer cells (ZHER2-ΔHBc). siRNA was encapsulated within the ZHER2-ΔHBc particles following disassembly and reassembly. The ZHER2-ΔHBc–siRNA hybrids were able to secure the encapsulated siRNA from serum and nucleases in vitro. Enhanced siRNA uptake in HER2-expressing cancer cells treated with ZHER2-ΔHBc–siRNA hybrids was observed compared to the nontargeted HBc–siRNA hybrids in a time- and dose-dependent manner. A successful in vitro polo-like kinase 1 (PLK1) gene knockdown was demonstrated in cancer cells treated with ZHER2-ΔHBc–siPLK1 hybrids, to levels comparable to commercial transfecting reagents. Interestingly, ZHER2-ΔHBc particles exhibit intrinsic capability of reducing the solid tumor mass, independent of siPLK1 therapy, in an intraperitoneal tumor model following intraperitoneal injection.

Tumor suppressor ZHX2 restricts hepatitis B virus replication via epigenetic and non-epigenetic manners

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), the stable genomic form as the template for viral transcription, plays a crucial role in viral persistence which remains a major global health problem. While accumulating evidence suggests the involvement of transcription factors and epigenetic machinery in cccDNA transcription, the roles of host transcription factors which contribute to epigenetic modification of cccDNA remain largely unknown. Zinc finger and homeoboxes 2 (ZHX2) is abundantly expressed in adult hepatocytes, where it acts as a transcriptional repressor and tumor suppressor by directly inhibiting the promoter activities of target genes. However, whether ZHX2 influences HBV replication or is involved in cccDNA epigenetic regulation remain unknown. In this study, we investigated the role of ZHX2 in cccDNA transcription. Analysis of immunohistochemistry showed that ZHX2 nuclear expression negatively correlated with serum HBV DNA and HBeAg. Remarkably, ZHX2 significantly decreased HBV antigens expression, pregenomic RNA (pgRNA) and HBV core particle DNA production both in vitro and in mouse livers supporting HBV antigens expression and cccDNA transcription. Dual luciferase and cccDNA ChIP assays confirmed that ZHX2 could bind to cccDNA and transcriptionally inhibit HBV promoter activities. In addition, ZHX2 suppressed the expression of histone regulator genes, such as cccDNA bound p300/CBP, and led to epigenetic repression of cccDNA. These findings highlight the roles of a novel restriction factor, ZHX2, in modulating HBV replication via regulating HBV promoter activities and cccDNA modifications. This study furthers our understanding of HBV transcription from cccDNA and offers new insights on potential HBV therapy.

Yield Optimisation of Hepatitis B Virus Core Particles in E. coli Expression System for Drug Delivery Applications

An E. coli expression system offers a mean for rapid, high yield and economical production of Hepatitis B Virus core (HBc) particles. However, high-level production of HBc particles in bacteria is demanding and optimisation of HBc particle yield from E. coli is required to improve laboratory-scale productivity for further drug delivery applications. Production steps involve bacterial culture, protein isolation, denaturation, purification and finally protein assembly. In this study, we describe a modified E. coli based method for purifying HBc particles and compare the results with those obtained using a conventional purification method. HBc particle morphology was confirmed by Atomic Force Microscopy (AFM). Protein specificity and secondary structure were confirmed by Western Blot and Circular Dichroism (CD), respectively. The modified method produced ~3-fold higher yield and greater purity of wild type HBc particles than the conventional method. Our results demonstrated that the modified method produce a better yield and purity of HBc particles in an E. coli-expression system, which are fully characterised and suitable to be used for drug delivery applications.

Establishment of a fluorescent in situ hybridization assay for imaging hepatitis B virus nucleic acids in cell culture models

While chronic hepatitis B remains a global public health problem, the detailed spatiotemporal dynamics of the key molecular events leading to the multiplication and egress of hepatitis B virus (HBV) are still largely unclear. Previously, we developed a chromogenic in situ hybridization assay for detection of HBV RNA, DNA and covalently closed circular DNA in clinical liver biopsies. Here, we report the establishment of a fluorescent in situ hybridization method for the visualization of HBV RNA, HBV core particle DNA and intranuclear DNA in a tetracycline-inducible HBV replication system (HepAD38) and a de novo infection system (HepG2-NTCP). Using 3D-STORM (three-dimensional stochastic optical reconstruction microscopy), we were able to obtain images of HBV RNA and DNA with improved spatial resolution allowing in-depth analyses of key virological events within complex subcellular compartments. Taken together, these techniques should facilitate a deeper understanding of the molecular events of the HBV life cycle and shed new light on the intricate mechanisms of virus–host interactions.Emerging Microbes & Infections (2017) 6, e98; doi:10.1038/emi.2017.84; published online 8 November 2017

Engineering hepatitis B virus core particles for targeting HER2 receptors invitro and invivo.

Hepatitis B Virus core (HBc) particles have been studied for their potential as drug delivery vehicles for cancer therapy. HBc particles are hollow nano-particles of 30–34 nm diameter and 7 nm thick envelopes, consisting of 180–240 units of 21 kDa core monomers. They have the capacity to assemble/dis-assemble in a controlled manner allowing encapsulation of various drugs and other biomolecules. Moreover, other functional motifs, i.e. receptors, receptor binding sequences, peptides and proteins can be expressed. This study focuses on the development of genetically modified HBc particles to specifically recognise and target human epidermal growth factor receptor-2 (HER2)-expressing cancer cells, in vitro and in vivo, for future cancer therapy. The non-specific binding capacity of wild type HBc particles was reduced by genetic deletion of the sequence encoding arginine-rich domains. A specific HER2-targeting was achieved by expressing the ZHER2 affibodies on the HBc particles surface. In vitro studies showed specific uptake of ZHER2-ΔHBc particles in HER2 expressing cancer cells. In vivo studies confirmed positive uptake of ZHER2-ΔHBc particles in HER2-expressing tumours, compared to non-targeted ΔHBc particles in intraperitoneal tumour-bearing mice models. The present results highlight the potential of these nanocarriers in targeting HER2-positive metastatic abdominal cancer following intra-peritoneal administration.

Effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus

To explore the effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus (HBV) and its possible mechanism. The hepatocyte-derived cell line HepG2 was co-transfected with DAI siRNA and the HBV1.3 replicative plasmid PHY106, and the cells were divided into two experimental groups. Six hours later, total RNA was extracted from the first group of cells and expression of IFIT1 and IL-6 were detected by real-time RT-PCR. The second group of cells was incubated for 4 days, after which the cell supernatant was collected and the HBV surface antigen (HBsAg) and envelope antigen (HBeAg) were detected by ELISA. In addition, HBV core particles were extracted and applied to southern blot assay to detect the intracellular HBV replication intermediates (rcDNA, dlDNA and ssDNA). Next, the HepG2 cells were triple transfected with siRNA targeting the type I interferon pathway molecule TBK1 and DAI simultaneously and HBV1.3, after which HBV viral proteins were detected. Two-group comparisons were made using the independent sample t-test, and more-than-2-group comparisons were made using ANOVA. DAI gene expression was down-regulated in response to DAI siRNA transfection. Cells with down-regulated DAI showed inhibited HBV replication (in a dose-dependent manner), accompanied by reduced levels of HBsAg (0.0195+/-0.0050 vs. 0.3150+/-0.0200, P less than 0.05, t = 14.77) and HBeAg (0.0140+/-0.0040 vs. 0.01235+/-0.0135, P less than 0.05, t = 7.777). No effect of down-regulated DAI was observed for the expression of IFIT1 of IL-6. siRNA-mediated down-regulation of TBK1 and DAI simultaneously led to reduced expression of HBsAg and HBeAg. Down-regulation of DAI gene expression inhibited HBV replication and HBV protein expression, but the underlying mechanism was not related to the type I interferon or NF-kB signaling pathway.

Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant loop region of hepatitis B core antigen: Insertion of multiple copies of M2e increases immunogenicity and protective efficiency

The extracellular domain of the transmembrane protein M2 (M2e) of influenza A virus is a promising target for the development of “universal” vaccines against influenza. M2e is a poor immunogen by itself; however, when M2e is linked to an appropriate carrier, such as hepatitis B virus core (HBc) particles, it becomes highly immunogenic. Insertions of target peptides into the surface-exposed major immunodominant loop region (MIR) of the HBc antigen are especially immunogenic, but such insertions often affect the protein folding and formation of recombinant virus-like particles. To facilitate an appropriate conformation of the M2e insert, we introduced flexible linkers at the junction points between the insert and flanking HBc sequences. This approach allowed the construction of recombinant HBc particles carrying 1, 2 and 4 copies of M2e in the MIR region. These particles were produced in Escherichia coli and purified to homogeneity. The immune response and protective activity of hybrid HBc particles in mice correlated with the number of inserted M2e peptides: the highest immunogenicity and complete protection of mice against the lethal challenge by influenza virus was observed with particles carrying four copies of M2e. The possibility of the simultaneous presentation of M2e peptides from several important influenza strains on a single HBc particle could also facilitate the development of a broad-specificity vaccine efficient not only against influenza A strains of human origin but also for newly emerging strains of animal origin, such as the avian influenza.

Mutation of arginine residues to avoid non-specific cellular uptakes for hepatitis B virus core particles.

BackgroundThe hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150–159 in glycine (G) residues.ResultsThe mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells.ConclusionsBecause this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0074-8) contains supplementary material, which is available to authorized users.

Hepatitis B virus nucleocapsid particles produced in eukaryotic cells: Properties and purification

Hepatitis B virus nucleocapsid particles produced in eukaryotic cells: Properties and purification

Serum HBV RNA as a possible marker of HBV replication in the liver during nucleot(s)ide analogue therapy

We read with interest the article by Tsuge et al. [1] published in the recent issue of the Journal of Gastroenterology. Treatment with nucleot(s)ide analogue (NUC) strongly suppresses the replication of hepatitis B virus (HBV) leading to a high rate of serum HBV DNA negativity. However, the incidence of relapse after the cessation of NUCs is high. Criterion for safe discontinuation of NUC therapy after long term therapy is not established to date. In HBe antigen positive patients, seroconversion, HBV DNA negativity and consolidation therapy of[6 months may be a consensus criteria but 30–50 % of patients fulfilling this criteria experience a relapse. In HBe antigen negative patients, NUC therapy is generally recommended until HBs antigen becomes undetected. Tsuge et al. [1] measured serum HBV RNA plus DNA by real time PCR and showed that the serum HBV DNA ? RNA titer following 3 months of NUC treatment was a significant predictor of early (within 24 weeks) HBV DNA rebound after discontinuation of NUC. The serum HBV DNA ? RNA titer was also associated with ALT rebound in HBe antigen positive patients. The results of the study by Tsuge et al. indicate that serum HBV DNA ? RNA titer may serve as predictor of relapse after discontinuation of NUC. The high rate of relapse after discontinuation of NUC is due to the persistence of HBV replication in the liver even during the NUC therapy. The replicative intermediate form of HBV, covalently closed circular DNA (cccDNA), may not be eliminated by NUC therapy and serves as a template for viral pre-genomic messenger RNA [2]. This concept was proved by a study showing that quantification of intrahepatic HBV cccDNA had a high accuracy of predicting sustained virological response after NUC discontinuation [3]. Still, we need a non-invasive and clinically usable marker for the assessment of HBV replication in the liver during NUC therapy. The measurement of HBV core related antigen may be an alternative [4]. The rationale of measuring HBV RNA in serum was that immature HBV particles including HBV RNA are released from hepatocytes during NUC treatment under the circumstances that pre-genomic HBV RNA are transcribed from HBV cccDNA, packaged into HBV core particles, but not reverse transcribed into plus-strand HBV DNA due to strong interference by NUC, and the excessive amounts of these immature particles are accumulated in hepatocytes [5, 6]. Tsuge et al. showed that serum HBV DNA ? RNA titer following 3 months of NUC treatment was significantly lower in patients with no rebound of HBV DNA. By using a cut-off value of 4.8 log copies/mL, the cumulative incidence of HBV DNA rebound was significantly lower in patients with serum HBV DNA ? RNA titer \ 4.8 log at 3 months of NUC treatment. The same groups previously showed that HBV RNA levels at 3 months of lamivudine treatment were predictor of early emergence of resistant mutations [7]. Taken together, serum HBV DNA ? RNA titer may be linked to the level of HBV replication in the liver during NUC therapy. Monitoring of serum HBV DNA ? RNA response may be utilized in various decision makings in treatment of HBV patients with NUC therapy. Based on these important findings, several questions may remain for future elucidation. Commercially available transcription-mediated amplification and hybridization assay An answer to this letter to the editor is available at doi:10.1007/s00535-013-0801-6.