Precore Protein Research Articles

Diversity of HBV genotypes and their association with precore/basal core mutations among HBsAg-positive patients in Ibadan, Nigeria.

Background. Hepatitis B virus (HBV) is the most implicated cause of severe liver disease and hepatocellular carcinoma worldwide. Studies have shown that the basal core protein (BCP) and precore protein (PC) of HBV play a significant role in HBV-related carcinogenesis. There is a paucity of data on the type and effect of BCP and PC mutations in Nigeria. This study aims to genotype HBV and investigate any mutations within the BCP and PC among HBV patients in Ibadan, Nigeria. Methods. Forty HBV-DNA-positive patients were recruited into this study, and the viral load assay and genotyping by nested multiplex PCR were done. The partial X gene region was amplified and Sanger sequenced. The BPC and PC genomic regions were then analysed using bioinformatics. Results. Twenty-three participants recorded HBV DNA viral load of >20 000 IU, while 17 had <20 000 IU and 28 samples were genotyped. Five genotypes (A, B, C, D and E) and four mixed genotypes (AC, AD ACD and ABCD) were detected. Genotype AC was the most frequently encountered, while genotypes E and B were the least encountered. Mutation was highest in ages 34-45 years. Double mutation A1762T and G1764A within the BCP region was the most encountered mutation. Conclusions. We report a diverse HBV genetic landscape, with mixed infections between genotypes with BCP double-mutation A1762T/G1764A, signalling the likelihood of poor HBV-related liver disease prognosis. Our findings contribute to our understanding of the molecular characteristics of HBV and its potential implications for disease progression and management among HBV-infected Nigerians.

Novel fully automated prototype assays for specific detection of phosphorylated and non-phosphorylated Hepatitis B core antigens.

Hepatitis B core antigen (HBcAg) has been proposed as a surrogate marker to reflect transcriptional activity of HBV covalently closed circular DNA (cccDNA) during active infections and may be a valuable tool to monitor the efficacy of antiviral therapies. However, HBcAg-specific immunoassays are unavailable, and current assays that measure hepatitis B core-related antigen (HBcrAg) cannot distinguish between HBcAg, HBeAg, and precore (PreC) proteins. Two fully automated assays were developed to specifically detect phosphorylated HBcAg (P-HBcAg, representing non-HBV DNA-containing particles) and non-phosphorylated HBcAg (representing HBV DNA-containing particles) circulating in HBV infected patients. P-HBcAg and HBcAg levels were analyzed in 124 single timepoint patients with active infections, in three longitudinal specimens from patients with acute HBV infections, and in four chronic hepatitis B (CHB) patients on-therapy (TDF - tenofovir disoproxil fumarate, pegIFN - pegylated interferon, NAPs - nucleic acids polymers). Analyzing acute infections revealed that P-HBcAg and HBcAg levels correlate more closely than HBcrAg to HBV DNA. During antiviral treatment of CHB patients, HBcAg correlates well with HBV DNA and indicates a therapeutic response to the treatment at the beginning of the therapy. In contrast, P-HBcAg tracks more closely to HBV RNA. Importantly, P-HBcAg is detectable several months after HBcAg became undetectable indicating that cccDNA is still transcriptionally active in hepatocytes. Overall, the ability to specifically distinguish between the various states of HBcAg (phosphorylated and non-phosphorylated) can provide additional insights for disease staging, drug development, and management of HBV therapies.

Diagnostic Performance of Precore Protein 22 Kilodalton Levels of HBV DNA in Chronic Hepatitis B Patients

Hepatitis B Virus (HBV) infection causes inflammation of the liver, which has a high prevalence in both Indonesia and the world. Serum HBV deoxyribonucleic acid (DNA) is important in determining the initiation therapy for Chronic Hepatitis B (CHB) patients. However, it has several limitations. Precore protein 22 kilodalton (p22cr) is synthesized from the HBV gene in hepatocytes, representing covalently closed circle (ccc) DNA. This study aimed to analyze the diagnostic performance of p22cr levels on HBV DNA in CHB patients. An observational analytic study with a cross-sectional approach was conducted on 83 CHB patients who were examined at the Clinical Pathology Laboratory of Dr. Moewardi General Academic Hospital in December 2020. Blood plasma samples were taken for HBV DNA and p22cr examination by using Polymerase Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA), respectively. The cut-off level of p22cr was determined by the Receiver Operating Curve (ROC) with the widest area Under the Curve (AUC). Sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Likelihood Ratio (PLR), Negative Likelihood Ratio (NLR), and accuracy were calculated for the diagnostic performance of p22cr. The cut-off point of p22cr on HBV DNA &gt; 20,000 IU/mL was 7.440 ng/mL with AUC 0.693 (p=0.003). The diagnostic performance of p22cr levels on HBV DNA obtained 44.44% sensitivity, 82.98% specificity, 66.67% PPV, 66.10% NPV, 2.61 PLR, 0.67 NLR, and 66.27% accuracy. P22cr level has a good specificity so it can be an alternative examination of HBV DNA in making decisions on therapy in patients with chronic hepatitis B. Further research needs to be done using HBcrAg and excluding elderly patients.

Characterization of Intracellular Precore-Derived Proteins and Their Functions in Hepatitis B Virus-Infected Human Hepatocytes.

Hepatitis B virus (HBV) precore protein is not essential for viral replication but is thought to facilitate chronic infection. In addition to the secreted precore products, including the hepatitis B e antigen (HBeAg) and PreC protein, intracellular precore-derived proteins in HBV-infected human hepatocytes remain poorly characterized, and their roles, if any, remain largely unknown. Here, we detected multiple precore derivatives, including the nonprocessed precursor p25 and the processing intermediate p22, in HBV-infected human hepatocytes as well as human hepatoma cells overexpressing the HBV precore protein. Both p25 and p22 showed phosphorylated and unphosphorylated forms, which were located in different intracellular compartments. Interestingly, precore expression was associated with decreases in intracellular HBV core protein (HBc) and secreted DNA-containing virions but was also associated with an increase in secreted empty virions. The decrease in HBc by precore could be attributed to cytosolic p22, which caused HBc degradation, at least in part by the proteasome, and consequently decreased HBV pregenomic RNA packaging and DNA synthesis. In addition, cytosolic p22 formed chimeric capsids with HBc in the cell, which were further secreted in virions. In contrast, the PreC antigen, like HBeAg, was secreted via the endoplasmic reticulum (ER)-Golgi secretory pathway and was thus unable to form capsids in the cell or be secreted in virions. Furthermore, p25, as well as p22, were secreted in virions from HBV-infected human hepatocytes and were detected in the sera of HBV-infected chimpanzees. In summary, we have detected multiple intracellular precore-derived proteins in HBV-infected human hepatocytes and revealed novel precore functions in the viral life cycle. IMPORTANCE Chronic hepatitis B remains a worldwide public health issue. The hepatitis B virus (HBV) precore protein is not essential for HBV replication but may facilitate viral persistence. In this study, we have detected multiple precore protein species in HBV-infected human hepatocytes and studied their functions in the HBV life cycle. We found that the HBV precore proteins decreased intracellular HBV core protein and reduced secretion of complete virions but enhanced secretion of empty virions. Interestingly, the cytosolic precore protein species formed chimeric capsids with the core protein and were secreted in virions. Our results shed new light on the functions of intracellular precore protein species in the HBV life cycle and have implications for the roles of precore proteins in HBV persistence and pathogenesis.

A critique and systematic review of the clinical utility of hepatitis B core-related antigen

Hepatitis B core-related antigen (HBcrAg) is a new biomarker for chronic hepatitis B (CHB) whose performance has not been critically or systematically appraised. Herein, we performed a systematic review to determine its clinical utility. We evaluated the biological pathway of HBcrAg and performed a systematic review of PubMed for clinical trials, cohort studies, and case-control studies that evaluated the clinical utility of HBcrAg. The effectiveness of HBcrAg in predicting HBV-specific clinical events (e.g. HBeAg seroconversion, phases of CHB, HBsAg loss, treatment response, and relapse after stopping therapy) was examined using receiver-operating characteristic curves. The correlation coefficients of HBcrAg with HBV DNA, quantitative HBsAg (qHBsAg), HBV RNA, and cccDNA were summarised from published studies. Median values were used as estimates. HBcrAg consists of three precore/core protein products: HBcAg, HBeAg, and a 22kDa precore protein. HBcrAg assays have been associated with false-positive rates of 9.3% and false-negative rates of between 12-35% for CHB. The new iTACT-HBcrAg is more sensitive but does not reduce the false-positive rate. A PubMed search found 248 papers on HBcrAg, of which 59 were suitable for analysis. The clinical performance of HBcrAg was evaluated using AUROC analyses, with median AUROCs of 0.860 for HBeAg seroconversion, 0.867 for predicting HBeAg(-) hepatitis, 0.645 for HBsAg loss, 0.757 for treatment response, and 0.688 for relapse after stopping therapy. The median correlation coefficient (r) was 0.630 with HBV DNA, 0.414 with qHBsAg, 0.619 with HBV RNA and 0.550 with cccDNA. Correlation decreased during antiviral therapy, but combined biomarkers improved performance. HBcrAg has a mixed performance and has a poor correlation with HBsAg loss and antiviral therapy, hence HBcrAg results should be interpreted with caution. Hepatitis B core-related antigen (HBcrAg) has been used to assess management of patients with chronic hepatitis B (CHB) without a systematic and critical Sreview of its performance. Our finding that HBcrAg had a false-positive rate of 9% and a false-negative rate of 12-35% raises concerns, although larger studies are needed for validation. A systematic review showed that the performance of HBcrAg was variable depending on the CHB endpoint; it was excellent at predicting HBeAg seroconversion and HBeAg-negative chronic hepatitis (vs. chronic infection), which should be its main use, but it was poor for relapse after stopping antiviral therapy and for HBsAg loss. HBcrAg results should be interpreted with considerable caution, particularly by physicians, researchers, guideline committees and agencies that approve diagnostic tests.

Constrained evolution of overlapping genes in viral host adaptation: Acquisition of glycosylation motifs in hepadnaviral precore/core genes.

Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes.

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.

Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis.

The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic “HAP” pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically “normal” capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.

Hepatitis B Core-Related Antigen: From Virology to Clinical Application.

Hepatitis B core-related antigen (HBcrAg) is a composite measure of the serum levels of hepatitis B e antigen, hepatitis B core antigen, and a 22-kDa precore protein. It has been shown to reflect the level and transcriptional activity of covalently closed circular DNA in the liver. Longitudinal cohort studies have improved our understanding of the role of this novel viral marker in the natural history of chronic hepatitis B. HBcrAg kinetics reflect the response to peginterferon, and its role in defining guidelines for stopping peginterferon therapy has been evaluated. HBcrAg is a marker of intrahepatic viral activity, which may influence the risk of hepatocellular carcinoma. In this article, we review the virology and role of HBcrAg in defining phases of chronic hepatitis B. Furthermore, the function of HBcrAg in predicting treatment outcomes and its role in monitoring response to novel antiviral agents will be discussed.

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.

Clinical efficacy of a novel, high-sensitivity HBcrAg assay in the management of chronic hepatitis B and HBV reactivation

A fully automated, novel high-sensitivity hepatitis B core-related antigen assay (iTACT-HBcrAg) has been developed. We demonstrate the clinical utility of iTACT-HBcrAg for monitoring chronic hepatitis B (CHB) and for the early detection of HBV reactivation. After fundamental assessments, the clinical performance of iTACT-HBcrAg was compared with other HBV markers. i) Serial sera, available from 161 HBeAg-negative patients with CHB and persistently undetectable HBV DNA, were measured by iTACT-HBcrAg and a conventional HBcrAg assay (G-HBcrAg). ii) Serial sera from 13 HBV-reactivated patients were measured by iTACT-HBcrAg and an ultra-high-sensitivity HBsAg immune complex transfer-chemiluminescent enzyme immunoassay (lower limit of detection; 0.0005IU/ml, ICT-CLEIA) to compare HBV DNA detection. iii) To elucidate the various HBcrAg components detected by iTACT-HBcrAg, OptiPrep density gradient centrifugation analysis was performed on sera obtained before and after HBV reactivation. The analytical performance of iTACT-HBcrAg was satisfactory. The sensitivity of iTACT-HBcrAg (2.1LogU/ml) was approximately 10-fold greater than that of G-HBcrAg (2.8LogU/ml). i) HBcrAg was detectable in the sera of 97.5% (157/161) of patients with CHB by iTACT-HBcrAg, of whom 75.2% (121/161) had ≥2.8LogU/ml HBcrAg and 22.4% (36/161) had 2.1-2.8LogU/ml HBcrAg, which was undetectable by G-HBcrAg. ii) 9 and 2 of 13 HBV-reactivated patients were HBcrAg-positive by iTACT-HBcrAg before and at HBV DNA positivity, respectively; 7 and 4 were HBcrAg-positive by iTACT-HBcrAg before and at HBsAg-positivity by ICT-CLEIA, respectively. iii) The HBcrAg detected by iTACT-HBcrAg before HBV reactivation was contained in empty particles (22KDa precore protein). iTACT-HBcrAg could be used to better monitor responses to anti-HBV treatments in HBeAg-negative patients and for the early detection of HBV reactivation. A fully automated, novel high-sensitivity hepatitis B core-related antigen assay (iTACT-HBcrAg) has been developed. iTACT-HBcrAg can be used to monitor HBeAg-negative patients with chronic hepatitis B, as well as for the early detection of HBV reactivation. iTACT-HBcrAg could be used as a general marker of disease progression and treatment response.

The Hepatitis B Virus Pre-Core Protein p22 Activates Wnt Signaling.

An emerging theme for Wnt-addicted cancers is that the pathway is regulated at multiple steps via various mechanisms. Infection with hepatitis B virus (HBV) is a major risk factor for liver cancer, as is deregulated Wnt signaling, however, the interaction between these two causes is poorly understood. To investigate this interaction, we screened the effect of the various HBV proteins for their effect on Wnt/β-catenin signaling and identified the pre-core protein p22 as a novel and potent activator of TCF/β-catenin transcription. The effect of p22 on TCF/β-catenin transcription was dose dependent and inhibited by dominant-negative TCF4. HBV p22 activated synthetic and native Wnt target gene promoter reporters, and TCF/β-catenin target gene expression in vivo. Importantly, HBV p22 activated Wnt signaling on its own and in addition to Wnt or β-catenin induced Wnt signaling. Furthermore, HBV p22 elevated TCF/β-catenin transcription above constitutive activation in colon cancer cells due to mutations in downstream genes of the Wnt pathway, namely APC and CTNNB1. Collectively, our data identifies a previously unappreciated role for the HBV pre-core protein p22 in elevating Wnt signaling. Understanding the molecular mechanisms of p22 activity will provide insight into how Wnt signaling is fine-tuned in cancer.

Monitoring of intracellular localization of Hepatitis B virus P22 protein using Laser Scanning Confocal Microscopy and Airyscan.

The aim of this study was to assess nucleo-cytoplasmic protein localization to better understand the exact intracellular localization of viral proteins involved with infections. Having determined the general protein localization of hepatitis B virus P22 precore protein, the aim was to more specifically resolve its intracellular organization. This was done using both laser scanning microscopy and Airyscan techniques. Using a 63× objective, the resolution obtained with Airyscan was increased by 1.5-fold as compared to confocal microscopy (p value <.00001).

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. Incontrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

Hepatitis B Virus Precore Protein p22 Inhibits Alpha Interferon Signaling by Blocking STAT Nuclear Translocation.

Antagonism of host immune defenses against hepatitis B virus (HBV) infection by the viral proteins is speculated to cause HBV persistence and the development of chronic hepatitis. The circulating hepatitis B e antigen (HBeAg, p17) is known to manipulate host immune responses to assist in the establishment of persistent viral infection, and HBeAg-positive (HBeAg+) patients respond less effectively to IFN-α therapy than do HBeAg-negative (HBeAg-) patients in clinical practice. However, the function(s) of the intracellular form of HBeAg, previously reported as the precore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we report that the cytosolic p22 protein, but not the secreted HBeAg, significantly reduces interferon-stimulated response element (ISRE) activity and the expression of interferon-stimulated genes (ISGs) upon alpha interferon (IFN-α) stimulation in cell cultures. In line with this, HBeAg+ patients exhibit weaker induction of ISGs in their livers than do HBeAg- patients upon IFN-α therapy. Mechanistically, while p22 does not alter the total STAT1 or pSTAT1 levels in cells treated with IFN-α, it blocks the nuclear translocation of pSTAT1 by interacting with the nuclear transport factor karyopherin α1 through its C-terminal arginine-rich domain. In summary, our study suggests that HBV precore protein, specifically the p22 form, impedes JAK-STAT signaling to help the virus evade the host innate immune response and, thus, causes resistance to IFN therapy.IMPORTANCE Chronic hepatitis B virus (HBV) infection continues to be a major global health concern, and patients who fail to mount an efficient immune response to clear the virus will develop a life-long chronic infection that can progress to chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma. There is no definite cure for chronic hepatitis B, and alpha interferon (IFN-α) is the only available immunomodulatory drug, to which only a minority of chronic patients are responsive, with hepatitis B e antigen (HBeAg)-negative patients responding better than HBeAg-positive patients. We herein report that the intracellular HBeAg, also known as precore or p22, inhibits the antiviral signaling of IFN-α, which sheds light on the enigmatic function of precore protein in shaping HBV chronicity and provides a perspective toward areas that need to be further studied to make the current therapy better until a cure is achieved.

Direct Inhibition of Hepatitis B e Antigen by Core Protein Allosteric Modulator.

Hepatitis B e antigen (HBeAg) is an important immunomodulator for promoting host immune tolerance during chronic hepatitis B (CHB) infection. In patients with CHB, HBeAg loss and seroconversion represent partial immune control of CHB infection and are regarded as valuable endpoints. However, the current approved treatments have only a limited efficacy in achieving HBeAg seroconversion in HBeAg‐positive patients. Hepatitis B virus (HBV) core protein has been recognized as an attractive antiviral target, and two classes of core protein allosteric modulator (CpAM) have been discovered: the phenylpropenamides (PPAs) and the heteroaryldihydropyrimidines (HAPs). However, their differentiation and potential therapeutic benefit beyond HBV DNA inhibition remain to be seen. Here, we show that in contrast to PPA series compound AT‐130, a HAP CpAM, HAP_R01, reduced HBeAg levels in multiple in vitro and in vivo HBV experimental models. Mechanistically, we found that HAP_R01 treatment caused the misassembly of capsids formed by purified HBeAg in vitro. In addition, HAP_R01 directly reduces HBeAg levels by inducing intracellular precore protein misassembly and aggregation. Using a HAP_R01‐resistant mutant, we found that HAP_R01‐mediated HBeAg and core protein reductions were mediated through the same mechanism. Furthermore, HAP_R01 treatment substantially reduced serum HBeAg levels in an HBV mouse model. Conclusion: Unlike PPA series compound AT‐130, HAP_R01 not only inhibits HBV DNA levels but also directly reduces HBeAg through induction of its misassembly. HAP_R01, as well as other similar CpAMs, has the potential to achieve higher anti‐HBeAg seroconversion rates than currently approved therapies for patients with CHB. Our findings also provide guidance for dose selection when designing clinical trials with molecules from HAP series.

The evaluation of fibrotic effects of the hepatitis B virus pre-core in hepatic stellate cells.

The role of the hepatitis B virus (HBV) endogenous pre-core protein in liver fibrosis is controversial. Whether the expression of the pre-core induces the activation of human stellate cells (HSCs) has not yet been reported. Plasmids expressing HBx, or pre-core protein were transfected into LX-2 cells. Subsequently, total RNA extracted and reverse transcription-quantitative polymerase chain reaction was performed to measure the fold change of collagen type I, α1 chain, α-smooth muscle actin and TIMP metalloproteinase inhibitor-1. Moreover, transforming growth factor (TGF)-β in the supernatant of HSCs was evaluated by ELISA assay. In addition, a MTT assay was performed to test the cytotoxicity of the endogenous expression in LX-2 cells. None of the plasmids exhibited cytotoxic nor significant proliferative effects on LX-2 cells by MTT assessment. The gene expression analysis of fibrotic genes in LX-2 cells demonstrated that the pre-core protein presented no significant (P>0.05) fibrotic impact when compared to the empty control plasmid and HBx. The data from the TGF-β ELISA was consistent with the mRNA expression as detected with the control plasmid (P>0.05). The endogenous expression of the HBV pre-core exhibited no fibrotic impression in HSCs when compared to HBx.

Predictive Value of Hepatitis B Core-Related Antigen (HBcrAg) During the Natural History of Hepatitis B Virus Infection.

The natural history of HBV infection includes immune tolerance (IT), immune clearance (IC), HBeAg-negative inactive/quiescent carrier (ENQ), and HBeAg-negative hepatitis (ENH) phases. As the current biomarkers for discriminating the four phases still have some weaknesses, additional serological indicators are needed. Hepatitis B core-related antigen (HBcrAg) encoded with the precore/core gene contains denatured HBeAg, HBV core antigen (HBcAg), and a 22-KDa precore protein (p22cr) and has been demonstrated to have a close association with the natural history of hepatitis B infection. However, no specific cutoff values and diagnostic parameters have been identified to evaluate the diagnostic efficacy. This study aimed to clarify the distribution of HBcrAg levels and evaluate the diagnostic performance during the natural history of HBV infection in a Western Chinese population. In this study, 294 samples were collected from treatment-naive HBV infection patients in different phases (IT = 64; IC = 72; ENQ = 100, and ENH = 58). We detected the HBcrAg values and analyzed the relationship between HBcrAg and HBV DNA. HBsAg and other clinical parameters were quantitatively detected. The HBcrAg levels of IT, IC, ENQ, and ENH were 9.30 log U/mL, 8.80 log U/mL, 3.00 log U/mL, and 5.10 log U/mL, respectively (p < 0.0001). Receiver operating characteristic curve analysis demonstrated that the areas under the curves (AUCs) for HBcrAg and quantitative HBsAg at cutoff values of 9.25 log U/mL and 4.355 log IU/mL for distinguishing the IT phase from the IC phase were 0.704 and 0.694, with a sensitivity of 53.13% and 79.69% and specificity of 76.39% and 59.72%, respectively. AUCs of HBcrAg and quantitative HBsAg at cutoff values of 4.15 log U/mL and 2.395 log IU/mL for discriminating between the ENQ and ENH phases were 0.931 and 0.653, with a sensitivity of 87.93% and 91.38% and specificity of 84.00% and 39.00%, respectively. HBcrAg levels varied significantly among the four natural phases of HBV infection and had higher predictive performance than quantitative HBsAg for distinguishing between ENQ-patients and ENH-patients and a similar performance as HBsAg for the discrimination between IT and IC phases, which indicated that HBcrAg could be a potential serological marker for HBV infection.

HBV secretion is regulated through the activation of endocytic and autophagic compartments mediated by Rab7 stimulation.

The cellular mechanisms by which hepatitis B virus (HBV) is assembled and exported are largely undefined. Recently, it has been suggested that these steps require the multivesicular body (MVB) and the autophagic machinery. However, the mechanisms by which HBV might regulate these compartments are unclear. In this study, we have found that by activating Rab7a, HBV alters its own secretion by inducing dramatic changes in the morphology of MVB and autophagic compartments. These changes are characterized by the formation of numerous tubules that are dependent upon the increase in Rab7 activity observed in the HBV-expressing HepG2.2.15 cells compared to HepG2 cells. Interestingly, transfection-based expression of the five individual viral proteins indicated that the precore protein, which is a precursor of HBeAg, was largely responsible for the increased Rab7 activity. Finally, small interfering RNA (siRNA)-mediated depletion of Rab7 significantly increased the secretion of virions, suggesting that reduced delivery of the virus to the lysosome facilitates viral secretion. These findings provide novel evidence indicating that HBV can regulate its own secretion through an activation of the endo-lysosomal and autophagic pathway mediated by Rab7 activation.

Conserved aromatic residues of the hepatitis B virus Precore propeptide are involved in a switch between distinct dimeric conformations and essential in the formation of heterocapsids

The Hepatitis B virus Precore protein, present in the secretory pathway as the HBeAg precursor, can associate in the cytoplasm with the Core protein to form heterocapsids, likely to favor viral persistence. Core and Precore proteins share their primary sequence except for ten additional aminoacids at the N-terminus of Precore. To address the role of this propeptide sequence in the formation of Precore heterocapsids, we designed a Precore mutant in which the two propeptide tryptophans are replaced by glycines. This mutant retains the properties of the wild-type Precore, notably cell trafficking and ability to interact with Core. However, it is not incorporated into heterocapsids and forms stable dimers distinct from the labile HBe dimers and the presumably Core-like dimers assembled into heterocapsids. Our data highlights the essential role of Precore׳s propeptide in switching between different conformations for different functions and pinpoint the propeptide Tryptophan residues as central in these properties.