Selective depletion of HBV-infected hepatocytes by class A capsid assembly modulators requires high levels of intrahepatic HBV core protein.

Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.

Hepatitis B virus core protein as a Rab-GAP suppressor driving liver disease progression.

Chronic hepatitis B virus (HBV) infection can lead to advanced liver pathology. Here, we establish a transgenic murine model expressing a basic core promoter (BCP)-mutated HBV genome. Unlike previous studies on the wild-type virus, the BCP-mutated HBV transgenic mice manifest chronic liver injury that culminates in cirrhosis and tumor development with age. Notably, agonistic anti-Fas treatment induces fulminant hepatitis in these mice even at a negligible dose. As the BCP mutant exhibits a striking increase in HBV core protein (HBc) expression, we posit that HBc is actively involved in hepatocellular injury. Accordingly, HBc interferes with Fis1-stimulated mitochondrial recruitment of Tre-2/Bub2/Cdc16 domain family member 15 (TBC1D15). HBc may also inhibit multiple Rab GTPase-activating proteins, including Rab7-specific TBC1D15 and TBC1D5, by binding to their conserved catalytic domain. In cells under mitochondrial stress, HBc thus perturbs mitochondrial dynamics and prevents the recycling of damaged mitochondria. Moreover, sustained HBc expression causes lysosomal consumption via Rab7 hyperactivation, which further hampers late-stage autophagy and substantially increases apoptotic cell death. Finally, we show that adenovirally expressed HBc in a mouse model is directly cytopathic and causes profound liver injury, independent of antigen-specific immune clearance. These findings reveal an unexpected cytopathic role of HBc, making it a pivotal target for HBV-associated liver disease treatment. The BCP-mutated HBV transgenic mice also provide a valuable model for understanding chronic hepatitis B progression and for the assessment of therapeutic strategies.

Precore mutation enhances viral replication to facilitate persistent infection especially in HBeAg-negative patients.

Naturally occurred precore (PC, G1896A) and/or basal core promoter (BCP, A1762T/G1764A) mutations are prevalent in chronic HBV-infected patients, especially those under HBeAg-negative status. However, the replicative capacity of HBV with PC/BCP mutations remains ambiguous. Herein, meta-analysis showed that, only under HBeAg-negative status, the serum HBV DNA load in patients with PC mutation was 7.41-fold higher than those without the mutation. Both PC mutation alone and BCP â€‹+ â€‹PC mutations promoted HBV replication in cell and hydrodynamic injection mouse models. In human hepatocyte chimeric mouse model, BCP â€‹+ â€‹PC mutations led to elevated replicative capacity and intrahepatic core protein accumulation. Mechanistically, preC RNA harboring PC mutation could serve as mRNA to express core and P proteins, and such pgRNA-like function favored the maintenance of cccDNA pool under HBeAg-negative status. Additionally, BCP â€‹+ â€‹PC mutations induced more extensive and severe human hepatocyte damage as well as activated endoplasmic reticulum stress and TNF signaling pathway in livers of chimeric mice. This study indicates that HBeAg-negative patients should be monitored on HBV mutations regularly and are expected to receive early antiviral treatment to prevent disease progression.

Class A capsid assembly modulator apoptotic elimination of hepatocytes with high HBV core antigen level in vivo is dependent on de novo core protein translation.

Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.

PreS1BP mediates inhibition of Hepatitis B virus replication by promoting HBx protein degradation.

BACKGROUND: PreS1-binding protein (PreS1BP), recognized as a nucleolar protein and tumor suppressor, influences the replication of various viruses, including vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1). Its role in hepatitis B virus (HBV) replication and the underlying mechanisms, however, remain elusive. METHODS: We investigated PreS1BP expression levels in an HBV-replicating cell and animal model and analyzed the impact of its overexpression on viral replication metrics. HBV DNA, covalently closed circular DNA (cccDNA), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and HBV RNA levels were assessed in HBV-expressing stable cell lines under varying PreS1BP conditions. Furthermore, co-immunoprecipitation and ubiquitination assays were used to detect PreS1BP- hepatitis B virus X protein (HBx) interactions and HBx stability modulated by PreS1BP. RESULTS: Our study revealed a marked decrease in PreS1BP expression in the presence of active HBV replication. Functional assays showed that PreS1BP overexpression significantly inhibited HBV replication and transcription, evidenced by the reduction in HBV DNA, cccDNA, HBsAg, HBcAg, and HBV RNA levels. At the molecular level, PreS1BP facilitated the degradation of HBx in a dose-dependent fashion, whereas siRNA-mediated knockdown of PreS1BP led to an increase in HBx levels. Subsequent investigations uncovered that PreS1BP accelerated HBx protein degradation via K63-linked ubiquitination in a ubiquitin-proteasome system-dependent manner. Co-immunoprecipitation assays further established that PreS1BP enhances the recruitment of the proteasome 20S subunit alpha 3 (PSMA3) for interaction with HBx, thereby fostering its degradation. CONCLUSIONS: These findings unveil a previously unidentified mechanism wherein PreS1BP mediates HBx protein degradation through the ubiquitin-proteasome system, consequentially inhibiting HBV replication. This insight positions PreS1BP as a promising therapeutic target for future HBV interventions. Further studies are warranted to explore the clinical applicability of modulating PreS1BP in HBV therapy.

A novel phthalazinone derivative as a capsid assembly modulator inhibits hepatitis B virus expression.

Development of new anti-hepatitis B virus (HBV) drugs that target viral capsid assembly is a very active research field. We identify a novel phthalazinone derivative, compound 5832, as a potent HBV inhibitor. In this study, we intend to elaborate the antiviral effect and mechanism of 5832 against HBV in vitro and in vivo. Compound 5832 treatment induces the formation of genome-free empty capsid by interfering with the core protein assembly domain, which significantly decreases the extracellular and intracellular HBV DNA. In the AAV-HBV transduced mouse model, 5832 suppresses serum HBV DNA after 4-week treatment, and decreases HBsAg and HBeAg levels. 5832 treatment also reduces intrahepatic HBV RNA, DNA and HBcAg levels. During the follow-up period after treatment withdrawal, serum antigen levels demonstrated no increase. We demonstrate 5832 treatment could active apoptotic signaling by elevating the expression of death receptor 5 (DR5), which participated in corresponding HBcAg-positive hepatocyte eradication. Phthalazinone derivative 5832 may serve as a promising anti-HBV drug candidate to improve the treatment options for chronic HBV infection.

Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids.

Effective process development towards intensified processing for gene delivery applications using Hepatitis B core Antigen (HBcAg) virus-like particles (VLPs) relies on analytical methods for the absolute quantification of HBcAg VLP proteins and bound nucleic acids. We investigated a silica spin column (SC)-based extraction procedure, including proteinase K lysis and silica chromatography, for the absolute quantification of different species of nucleic acids bound to HBcAg VLPs analyzed by dye-based fluorescence assays. This revealed load-dependent nucleic acid recoveries of the silica-SC-based extraction. We also developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method to separate and quantify the HBcAg proteins and the bound nucleic acids simultaneously without prior sample treatment by dissociation reagents. The method demonstrated sufficient linearity, accuracy, and precision coefficients and is suited for determining absolute protein and nucleic acid concentrations and HBcAg protein purities at various purification stages. Both the silica-SC-based extraction and the RP-based extraction presented overcome the limitations of analytical techniques, which are restricted to relative or qualitative analyses for HBcAg VLPs with bound nucleic acids. In combination with existing analytics, the methods for an absolute quantification of HBcAg VLPs and bound nucleic acids presented here are required to evaluate downstream purification steps, such as the removal of host cell-derived nucleic acids, concurrent protein loss, and efficient loading with therapeutic nucleic acids. Hence, the methods are key for effective process development when using HBcAg VLP as potential gene delivery vehicles.

High-level production of recombinant HBcAg virus-like particles in a mathematically modelled P. pastoris GS115 Mut+ bioreactor process under controlled residual methanol concentration.

Recombinant hepatitis B core antigen (HBcAg) molecules, produced in heterologous expression systems, self-assemble into highly homogenous and non-infectious virus-like particles (VLPs) that are under extensive research for biomedical applications. HBcAg production in the methylotrophic yeast P. pastoris has been well documented; however, productivity screening under various residual methanol levels has not been reported for bioreactor processes. HBcAg production under various excess methanol levels of 0.1, 1.0 and 2.0 g L-1 was investigated in this research. Results indicate that, under these particular conditions, the total process and specific protein yields of 876-1308 mg L-1 and 7.9-11.2 mg gDCW-1, respectively, were achieved after 67-75 h of cultivation. Produced HBcAg molecules were efficiently purified and the presence of highly immunogenic, correctly formed and homogenous HBcAg-VLPs with an estimated purity of 90% was confirmed by electron microscopy. The highest reported HBcAg yield of 1308 mg L-1 and 11.2 mg gDCW-1 was achieved under limiting residual methanol concentration, which is about 2.5 times higher than the next highest reported result. A PI-algorithm-based residual methanol concentration feed rate controller was employed to maintain a set residual methanol concentration. Finally, mathematical process models to characterise the vegetative, dead and total cell biomass (Xv, Xd and X), substrate (Glycerol and Methanol) concentration, reactor volume (V), and product (HBcAg) dynamics during cultivation, were identified. A rare attempt to model the residual methanol concentration during induction is also presented.

Conserved Lysine Residues of Hepatitis B Virus Core Protein Are Not Required for Covalently Closed Circular DNA Formation.

Hepatitis B virus (HBV) core protein (HBc), the building block of the viral capsid, plays a critical role throughout the HBV life cycle. There are two highly conserved lysine residues, namely, K7 and K96, on HBc, which have been proposed to function at various stages of viral replication, potentially through lysine-specific posttranslational modifications (PTMs). Here, we substituted K7 and K96 with alanine or arginine, which would also block potential PTMs on these two lysine residues, and tested the effects of these substitutions on HBV replication and infection. We found that the two lysine residues were dispensable for all intracellular steps of HBV replication. In particular, all mutants were competent to form the covalently closed circular DNA (cccDNA) via the intracellular amplification pathway, indicating that K7 and K96, or any PTMs of these residues, were not essential for nucleocapsid uncoating, a prerequisite for cccDNA formation. Furthermore, we found that K7A and K7R mutations did not affect de novo cccDNA formation and RNA transcription during infection, indicating that K7 or any PTMs of this residue were dispensable for HBV infection. In addition, we demonstrated that the HBc K7 coding sequence (AAA), as part of the HBV polyadenylation signal UAUAAA, was indispensable for viral RNA production, implicating this cis requirement at the RNA level, instead of any function of HBc-K7, likely constrains the identity of the 7th residue of HBc. In conclusion, our results provided novel insights regarding the roles of lysine residues on HBc, and their coding sequences, in the HBV life cycle. IMPORTANCE Hepatitis B virus (HBV) infection remains a public health burden that affects 296 million individuals worldwide. HBV core protein (HBc) is involved in almost all steps in the HBV life cycle. There are two conserved lysine residues on HBc. Here, we found that neither of them is essential for HBV intracellular replication, including the formation of covalently closed circular DNA (cccDNA), the molecular basis for establishing and sustaining the HBV infection. However, K96 is critical for virion morphogenesis, while the K7 coding sequence, but not HBc-K7 itself, is indispensable, as part of the RNA polyadenylation signal, for HBV RNA production from cccDNA. Our results provide novel insights regarding the role of the conserved lysine residues on HBc, and their coding sequences, in viral replication, and should facilitate the development of antiviral drugs against the HBV capsid protein.

N6-methyladenosine modification of the 5' epsilon structure of the HBV pregenome RNA regulates its encapsidation by the viral core protein.

Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5' end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m6A)-modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m6A modification of the 5' ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m6A site mutant in the HBV 5' epsilon, we present evidence that m6A methylation of 5' epsilon is necessary for its encapsidation. The m6A modification of 5' epsilon increased the efficiency of viral RNA packaging, whereas the m6A of 3' epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m6A modification at 5' epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5' epsilon m6A site-mutated pgRNA failed to interact. HBV polymerase interaction with 5' epsilon was independent of m6A modification of 5' epsilon. This study highlights yet another pivotal role of m6A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA-protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m6A modification.

Core Protein-Directed Antivirals and Importin ß Can Synergistically Disrupt Hepatitis B Virus Capsids.

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B virus (HBV) core protein (Cp) is involved in most stages of the viral life cycle; it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g., Impß) via Cp's C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp allosteric modulator (CpAM), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV life cycle is poorly understood. We investigate how HAP12 influences the interactions between empty or RNA-filled capsids with Impß and trypsin in vitro. We show that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrate that Impß synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize the disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resist the destabilizing effects of HAP12 and Impß. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV life cycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core protein-directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has the potential to disrupt the virus life cycle and activate the innate immune system.

Binding of a Pocket Factor to Hepatitis B Virus Capsids Changes the Rotamer Conformation of Phenylalanine 97.

(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses.

Blood-Based Biomarkers in Hepatitis B Virus-Related Hepatocellular Carcinoma, Including the Viral Genome and Glycosylated Proteins.

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) development and is a global public health issue. High performance biomarkers can aid the early detection of HCC development in HBV-infected individuals. In addition, advances in the understanding of the pathogenesis of HBV infection and in clinical laboratory techniques have enabled the establishment of disease-specific tests, prediction of the progression of liver diseases, including HCC, and auxiliary diagnosis of HCC, using blood-based methods instead of biopsies of liver or HCC tissues. Viral factors such as the HBV genotype, HBV genetic mutations, HBV DNA, and HBV-related antigens, as well as host factors, such as tumor-associated proteins and post-translational modifications, especially glycosylated proteins, can be blood-based, disease-specific biomarkers for HCC development in HBV-infected patients. In this review, we describe the clinical applications of viral biomarkers, including the HBV genome and glycosylated proteins, for patients at a risk of HBV-related HCC, based on their molecular mechanisms. In addition, we introduce promising biomarker candidates for practical use, including colony stimulating factor 1 receptor (CSF1R), extracellular vesicles, and cell-free, circulating tumor DNA. The clinical use of such surrogate markers may lead to a better understanding of the risk of disease progression and early detection of HCC in HBV-infected patients, thereby improving their prognosis.

In vitro expression of precore proteins of hepatitis B virus subgenotype A1 is affected by HBcAg, and can affect HBsAg secretion.

HBeAg, a non-particulate protein of hepatitis B virus (HBV), is translated from the precore/core region as a precursor, which is post-translationally modified. Subgenotype A1 of HBV, which is a risk factor for hepatocellular carcinoma (HCC), has unique molecular characteristics in the basic core promoter/precore regions. Carriers of A1 exhibit early HBeAg loss. We sought to further characterize the precore proteins of A1 in vitro. HuH-7 cells were transfected with subgenomic constructs expressing individual precore proteins. Western blot analysis using DAKO anti-core antibody showed the expected sizes and a 1 kDa larger band for P22, P20 and P17. Using confocal microscopy, a cytoplasmic accumulation of HBeAg and precursors was observed with P25-expressing plasmid, whereas P22 localized both in the cytoplasm and nucleus. P20 and P17, which lack the carboxy end of P22 showed strong nuclear accumulation, implicating a nuclear localization signal in the N-terminal 10 amino acids. G1862T, unique to subgenotype A1, is frequently found in HBV from HCC patients. P25 with G1862T showed delayed and reduced HBeAg expression/secretion. Knock-out of core in the replication competent clones led to precore protein accumulation in the cytoplasm/perinuclear region, and decreased HBeAg secretion. Knock-out of precore proteins increased HBsAg secretion but intracellular HBsAg expression was unaffected. Over-expression of precore proteins in trans led to decreased HBsAg expression and secretion. Intracellular trafficking of HBV A1 precore proteins was followed. This was unaffected by the CMV promoter and different cell types. In the viral context, precore protein expression was affected by absence of core, and affected HBsAg expression, suggesting an interrelationship between precore proteins, HBcAg and HBsAg. This modulatory role of HBeAg and its precursors may be important in viral persistence and ultimate development of HCC.

Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection.

BACKGROUND AND AIMS: The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints. APPROACH AND RESULTS: Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA. CONCLUSIONS: In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.

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.

HBV upregulated triggering receptor expressed on myeloid cells-1 (TREM-1) expression on monocytes participated in disease progression through NF-Kb pathway.

The triggering receptor expressed on myeloid cells-1 (TREM-1) signal is related to the continuous amplification of inflammatory pathway. However, it is not clear whether and how HBV can regulated the expression of TREM-1 on monocyte participated in the progression of liver disease. Here, we showed that the expression of TREM-1 on monocyte subsets were increased significantly in HBV related liver cirrhosis group compared with chronic infected group and healthy control group. HBsAg and HBeAg could up-regulated TREM-1 on monocyte by NF-KB pathway, and at least last for 72 h. Increased TREM-1 on monocyte might associated with high level of inflammatory cytokine (TNF-a, IL-1ß and IL-6) and the activation of LX-2 cells. Bioinformatics analysis showed that the high expression of TREM-1 was related to the poor prognosis of hepatocellular carcinoma (HCC). The level of TREM-1 might help to predict the progression of HBV infected liver disease and treat target to prevent fibrosis progression.

Covalent protein display on Hepatitis B core-like particles in plants through the in vivo use of the SpyTag/SpyCatcher system.

Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.

Intracellular Trafficking of HBV Particles.

The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles.

Distinct disease features in chimpanzees infected with a precore HBV mutant associated with acute liver failure in humans.

Transmission to chimpanzees of a precore hepatitis B virus (HBV) mutant implicated in acute liver failure (ALF) in humans did not cause ALF nor the classic form of acute hepatitis B (AHB) seen upon infection with the wild-type HBV strain, but rather a severe AHB with distinct disease features. Here, we investigated the viral and host immunity factors responsible for the unusual severity of AHB associated with the precore HBV mutant in chimpanzees. Archived serial serum and liver specimens from two chimpanzees inoculated with a precore HBV mutant implicated in ALF and two chimpanzees inoculated with wild-type HBV were studied. We used phage-display library and next-generation sequencing (NGS) technologies to characterize the liver antibody response. The results obtained in severe AHB were compared with those in classic AHB and HBV-associated ALF in humans. Severe AHB was characterized by: (i) the highest alanine aminotransferase (ALT) peaks ever seen in HBV transmission studies with a significantly shorter incubation period, compared to classic AHB; (ii) earlier HBsAg clearance and anti-HBs seroconversion with transient or undetectable hepatitis B e antigen (HBeAg); (iii) limited inflammatory reaction relative to hepatocellular damage at the ALT peak with B-cell infiltration, albeit less extensive than in ALF; (iv) detection of intrahepatic germline antibodies against hepatitis B core antigen (HBcAg) by phage-display libraries in the earliest disease phase, as seen in ALF; (v) lack of intrahepatic IgM anti-HBcAg Fab, as seen in classic AHB, but at variance with ALF; and (vi) higher proportion of antibodies in germline configuration detected by NGS in the intrahepatic antibody repertoire compared to classic AHB, but lower than in ALF. This study identifies distinct outcome-specific features associated with severe AHB caused by a precore HBV mutant in chimpanzees, which bear closer resemblance to HBV ALF than to classic AHB. Our data suggest that precore HBV mutants carry an inherently higher pathogenicity that, in addition to specific host factors, may play a critical role in determining the severity of acute HBV disease.