Hepatitis B Virus Gene Expression Research Articles

A mutual regulatory loop between transcription factor Yin Yang 1 and hepatitis B virus replication influences chronic hepatitis B

Hepatitis B virus (HBV) infections pose a major threat to human health. HBV can upregulate the expression of the transcription factor Yin Yang 1 (YY1) in in vitro cytological experiments, suggesting an association between YY1 and HBV infection. However, data on YY1 expression in chronic hepatitis B (CHB) patients are lacking. In this study, we aimed to assess the correlation between YY1 expression and HBV infection. We detected serum YY1 levels in 420 patients with chronic HBV infection, 30 patients with chronic hepatitis C virus infection, and 32 healthy controls using an enzyme-linked immunosorbent assay. The correlation between YY1 levels and clinical parameters was analyzed. Meanwhile, the changes of YY1 before and after interferon or entecavir treatment were analyzed. YY1 levels in the liver tissues were detected using immunofluorescence staining. The expression of YY1 in HBV-expressing cells was detected through western blotting. Meanwhile, we explored the effects of YY1 on HBV replication and gene expression. We found that YY1 was highly expressed in the serum and liver tissues of CHB patients. Serum YY1 levels positively correlated with HBV DNA and hepatitis B surface antigen (HBsAg). Additionally, HBV DNA levels increased but HBsAg levels decreased after HBV-expressing cells overexpress YY1. In conclusion, our study demonstrates that YY1 plays an important role in HBV replication and gene expression, providing a potential target for the treatment of CHB.

The lack of HBsAg secretion does neither facilitate induction of antiviral T cell responses nor Hepatitis B Virus clearance in mice

Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts is regarded as a key contributor. HBsAg is expressed in HBV-infected hepatocytes and those carrying an HBV integration. Whether either HBsAg secretion or the high antigen amount expressed in the liver determines its immunomodulatory properties, however, remains unclear. We, therefore, developed a novel HBV animal model that allowed us to study the role of secreted HBsAg. We introduced a previously described HBs mutation, C65S, abolishing HBsAg secretion into a replication-competent 1.3-overlength HBV genome and used adeno-associated virus vectors to deliver it to the mouse liver. The AAV-HBV established a carrier state of wildtype and C65S mutant HBV, respectively. We investigated antiviral B- and T-cell immunity in the HBV-carrier mice after therapeutic vaccination. Moreover, we compared the effect of a lacking HBsAg secretion with that of an antiviral siRNA. While missing HBsAg secretion allowed for higher levels of detectable anti-HBs antibodies after therapeutic vaccination, it did neither affect antiviral T-cell responses nor intrahepatic HBV gene expression, irrespective of the starting level. A treatment with HBV siRNA restricting viral antigen expression within hepatocytes, however, improved the antiviral efficacy of therapeutic vaccination, irrespective of the ability of HBV to secrete HBsAg. Our data indicate that clearing HBsAg from blood cannot significantly impact HBV persistence or T-cell immunity. This indicates that a restriction of hepatic viral antigen expression will be required to break HBV immunotolerance.

MiR-HCC2 suppresses hepatitis B virus replication by inhibiting the activity of the enhancer I/X promoter.

miR-HCC2 has been reported to markedly promote the growth, metastasis, and stemness of hepatocellular carcinoma (HCC) cells in vitro and in vivo. Deep sequencing showed that miR-HCC2 was significantly upregulated in hepatitis B virus (HBV)-positive (HBV+) HCC tissue samples compared with HBV-negative (HBV-) HCC tissue samples. miR-HCC2 expression was further evaluated in HCC tissues and cells, and the expression of miR-HCC2 was found to be significantly higher in HBV+ HCC tissues and cells than in HBV- HCC tissues and cells, suggesting that high miR-HCC2 expression could be induced by HBV infection. To explore the relationship between miR-HCC2 and HBV, we investigated the effect of miR-HCC2 on HBV antigen expression, transcription, and replication. We found that miR-HCC2 was involved in the negative feedback regulation of HBV replication. Further mechanistic studies revealed that miR-HCC2 suppressed HBV replication by inhibiting the activity of the enhancer I/Xpromoter. Our study demonstrates the effect of the inhibition of miR-HCC2 on HBV gene expression and replication, which can help to illustrate the complex regulatory network involving host miRNAs and HBV.

Deficiency of SCAP inhibits HBV pathogenesis via activation of the interferon signaling pathway

Hepatitis B virus (HBV) infects the liver and is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Approaches for an effective cure are thwarted by limited knowledge of virus-host interactions. Herein, we identified SCAP as a novel host factor that regulates HBV gene expression. SCAP, sterol regulatory element-binding protein (SREBP) cleavage-activating protein, is an integral membrane protein located in the endoplasmic reticulum. The protein plays a central role in controlling lipid synthesis and uptake by cells. We found that gene silencing of SCAP significantly inhibited HBV replication; furthermore, knockdown of SREBP2 but not SREBP1, the downstream effectors of SCAP, reduced HBs antigen production from HBV infected primary hepatocytes. We also demonstrated that knockdown of SCAP resulted in activation of interferons (IFNs) and IFN stimulated genes (ISGs). Conversely, ectopic expression of SREBP2 in SCAP-deficient cells restored expression of IFNs and ISGs. Importantly, expression of SREBP2 restored HBV production in SCAP knockdown cells, suggesting that SCAP participates in HBV replication through an effect on IFN production via its downstream effector SREBP2. This observation was further confirmed by blocking IFN signaling by an anti-IFN antibody, which restored HBV infection in SCAP-deficient cells. This led to the conclusion that SCAP regulates the IFN pathway through SREBP, thereby affecting the HBV lifecycle. This is the first study to reveal the involvement of SCAP in regulation of HBV infection. These results may facilitate development of new antiviral strategies against HBV.

Fibroblast Growth Factor 11 Inhibits Hepatitis B Virus Gene Expression Through FXRα Suppression

Fibroblast growth factor 11 (FGF11) is a member of the intracellular FGF family, which shows different signal transmission compared with other FGF superfamily members. The molecular function of FGF11 is not clearly understood. In this study, we identified the inhibitory effect of FGF11 on hepatitis B virus (HBV) gene expression through transcriptional suppression. FGF11 decreased the mRNA and protein expression of HBV genes in liver cells. While the nuclear receptor FXRα1 increased HBV promoter transactivation, FGF11 decreased the FXRα-mediated gene induction of the HBV promoter by the FXRα agonist. Reduced endogenous levels of FXRα by siRNA and the dominant negative mutant protein (aa 1–187 without ligand binding domain) of FXRα expression indicated that HBV gene suppression by FGF11 is dependent on FXRα inhibition. In addition, FGF11 interacts with FXRα protein and reduces FXRα protein stability. These results indicate that FGF11 inhibits HBV replicative expression through the liver cell-specific transcription factor, FXRα, and suppresses HBV promoter activity. Our findings may contribute to the establishment of better regimens for the treatment of chronic HBV infections by including FGF11 to alter the bile acid mediated FXR pathway.

Pre- and Post-Transcriptional Control of HBV Gene Expression: The Road Traveled towards the New Paradigm of HBx, Its Isoforms, and Their Diverse Functions.

Hepatitis B virus (HBV) is an enveloped DNA human virus belonging to the Hepadnaviridae family. Perhaps its main distinguishable characteristic is the replication of its genome through a reverse transcription process. The HBV circular genome encodes only four overlapping reading frames, encoding for the main canonical proteins named core, P, surface, and X (or HBx protein). However, pre- and post-transcriptional gene regulation diversifies the full HBV proteome into diverse isoform proteins. In line with this, hepatitis B virus X protein (HBx) is a viral multifunctional and regulatory protein of 16.5 kDa, whose canonical reading frame presents two phylogenetically conserved internal in-frame translational initiation codons, and which results as well in the expression of two divergent N-terminal smaller isoforms of 8.6 and 5.8 kDa, during translation. The canonical HBx, as well as the smaller isoform proteins, displays different roles during viral replication and subcellular localizations. In this article, we reviewed the different mechanisms of pre- and post-transcriptional regulation of protein expression that take place during viral replication. We also investigated all the past and recent evidence about HBV HBx gene regulation and its divergent N-terminal isoform proteins. Evidence has been collected for over 30 years. The accumulated evidence simply strengthens the concept of a new paradigm of the canonical HBx, and its smaller divergent N-terminal isoform proteins, not only during viral replication, but also throughout cell pathogenesis.

A spatiotemporally controlled recombinant cccDNA mouse model for studying HBV and developing drugs against the virus

Covalently closed circular (ccc) DNA is the template for hepatitis B virus (HBV) replication. The lack of small animal models for characterizing chronic HBV infection has hampered research progress in HBV pathogenesis and drug development. Here, we generated a spatiotemporally controlled recombinant cccDNA (rcccDNA) mouse model by combining Cre/loxP-mediated DNA recombination with the liver-specific “Tet-on/Cre” system. The mouse model harbors three transgenes: a single copy of the HBV genome (integrated at the Rosa26 locus, RHBV), H11-albumin-rtTA (spatiotemporal conditional module), and (tetO)7-Cre (tetracycline response element), and is named as RHTC mouse. By supplying the RHTC mice with doxycycline (DOX)-containing drinking water for two days, the animals generate rcccDNA in hepatocytes, and the rcccDNA supports active HBV gene expression and can maintain HBV viremia persistence for over 60 weeks. Persistent HBV gene expression induces intrahepatic inflammation, fibrosis, and dysplastic pathology, which closely mirrors the disease progression in clinical patients. Bepirovirsen, an antisense oligonucleotide (ASO) targeting all HBV RNA species, showed dose-dependent antiviral effects in the RHTC mouse model. The spatiotemporally controlled rcccDNA mouse is convenient and reliable, providing versatile small animal model for studying cccDNA-centric HBV biology as well as evaluating antiviral therapeutics.

Inhibition of HBV replication by EVA1A via enhancing cellular degradation of HBV components and its potential therapeutic application

Hepatitis B virus (HBV) DNA is much higher during HBeAg-positive chronic HBV infection (EP-CBI) than during HBeAg-negative chronic HBV infection (EN-CBI), although the necroinflammation in liver is minimal and the adaptive immune response is similar in both phases. We previously reported that mRNA levels of EVA1A were higher in EN-CBI patients. In this study, we aimed to investigate whether EVA1A inhibits HBV gene expression and examine the underlying mechanisms. The available cell models for HBV replication and model HBV mice were used to investigate how EVA1A regulates HBV replication and the antiviral activity based on gene therapy. The signaling pathway was determined through RNA sequencing analysis. The results demonstrated that EVA1A can inhibit HBV gene expression in vitro and in vivo. In particular, EVA1A overexpression resulted in accelerated HBV RNA degradation and activation of the PI3K-Akt-mTOR pathway, two processes that directly and indirectly inhibiting HBV gene expression. EVA1A is a promising candidate for treating chronic hepatitis B (CHB). In conclusion, EVA1A is a new host restriction factor that regulates the HBV life cycle via a nonimmune process.

Mitochondrial HIGD1A inhibits hepatitis B virus transcription and replication through the cellular PNKD-NF-κB-NR2F1 nexus.

Hepatitis B Virus (HBV) replication has been reported to be restricted by the intrahepatic host restriction factors and antiviral signaling pathways. The intracellular mechanisms underlying the significant viremia difference among different phases of the natural history chronic HBV infection remain elusive. We herein report that the hypoxia-induced gene domain protein-1a (HIGD1A) was highly expressed in the liver of inactive HBV carriers with low viremia. Ectopic expression of HIGD1A in hepatocyte-derived cells significantly inhibited HBV transcription and replication in a dose-dependent manner, while silence of HIGD1A promoted HBV gene expression and replication. Similar results were also observed in both de novo HBV-infected cell culture model and HBV persistence mouse model. Mechanistically, HIGD1A is located on the mitochondrial inner membrane and activates nuclear factor kappa B (NF-κB) signaling pathway through binding to paroxysmal nonkinesigenic dyskinesia (PNKD), which further enhances the expression of a transcription factor NR2F1 to inhibit HBV transcription and replication. Consistently, knockdown of PNKD or NR2F1 and blockage of NF-κB signaling pathway abrogated the inhibitory effect of HIGD1A on HBV replication. Mitochondrial HIGD1A exploits the PNKD-NF-κB-NR2F1 nexus to act as a host restriction factor of HBV infection. Our study thus shed new lights on the regulation of HBV by hypoxia-related genes and related antiviral strategies.

A RNase P Ribozyme Inhibits Gene Expression and Replication of Hepatitis B Virus in Cultured Cells.

Hepatitis B virus (HBV), an international public health concern, is a leading viral cause of liver disease, such as hepatocellular carcinoma. Sequence-specific ribozymes derived from ribonuclease P (RNase P) catalytic RNA are being explored for gene targeting applications. In this study, we engineered an active RNase P ribozyme, M1-S-A, targeting the overlapping region of HBV S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA), all deemed essential for viral infection. Ribozyme M1-S-A cleaved the S mRNA sequence efficiently in vitro. We studied the effect of RNase P ribozyme on HBV gene expression and replication using the human hepatocyte HepG2.2.15 culture model that harbors an HBV genome and supports HBV replication. In these cultured cells, the expression of M1-S-A resulted in a reduction of more than 80% in both HBV RNA and protein levels and an inhibition of about 300-fold in the capsid-associated HBV DNA levels when compared to the cells that did not express any ribozymes. In control experiments, cells expressing an inactive control ribozyme displayed little impact on HBV RNA and protein levels, and on capsid-associated viral DNA levels. Our study signifies that RNase P ribozyme can suppress HBV gene expression and replication, implying the promise of RNase P ribozymes for anti-HBV therapy.

The role of HBV cccDNA in occult hepatitis B virus infection.

Occult hepatitis B virus (HBV) infection (OBI) refers to the presence of replication-competent HBV DNA in the liver, with or without HBV DNA in the blood, in individuals who tested negative for HBV surface antigen (HBsAg). In this peculiar phase of HBV infection, the covalently closed circular DNA (cccDNA) is in a low state of replication. Several advances have been made toward clarifying the mechanisms involved in such a suppression of viral activity, which seems to be mainly related to the host's immune control and epigenetic factors. Although the underlying mechanisms describing the genesis of OBI are not completely known, the presence of viral cccDNA, which remains in a low state of replication due to the host's strong immune suppression of HBV replication and gene expression, appears to be the causative factor. Through this review, we have provided an updated account on the role of HBV cccDNA in regulating OBI. We have comprehensively described the HBV cell cycle, cccDNA kinetics, current regulatory mechanisms, and the therapeutic methods of cccDNA in OBI-related diseases.

The antiviral activity of tripartite motif protein 38 in hepatitis B virus replication and gene expression and its association with treatment responses during PEG-IFN-α antiviral therapy

Hepatitis B virus (HBV) infection represents one of the most critical health problems worldwide. Tripartite motif protein 38 (TRIM38) is an interferon-stimulated gene (ISG) that inhibits various DNA and RNA viruses.In this study, we found a mechanistic correlation between TRIM38 expression levels and the efficacy of HBV infection and IFN-α therapy in patients with CHB. TRIM38 was highly induced by IFN-alpha (IFN-α) in vivo and in vitro. TRIM38 overexpression inhibited HBV replication and gene expression in HepG2 and HepG2.2.15 cells, whereas knockdown of TRIM38 increased these processes. Further experiments indicated that TRIM38 protein enhanced the antiviral effect of IFN-α by enhancing the expression of antiviral proteins. A prospective study revealed high TRIM38 levels in peripheral blood PBMCs were from early responders, and increased TRIM38 expression correlated with a better response to PEG-IFN-α therapy. Taken together, our study suggests that TRIM38 plays a vital role in HBV replication and gene expression.

HBV Genomic Integration and Hepatocellular Carcinoma

The infection of hepatitis B virus (HBV) is one of the most important risk factors of liver carcinogenesis. The infection could influence several aspects of the host liver cells, including liver inflammation resulting in chronic hepatitis, cirrhosis, and even hepatocellular carcinoma (HCC). Among of them, HBV DNA integration into the host genome could change host gene expression profiling, genomic stability, and HBV gene expression. The present review is focused on the effects of the integration of HBV genomic DNA into host genome during the carcinogenesis of HCC.

Molecular clones of genetically distinct hepatitis B virus genotypes reveal distinct host and drug treatment responses

Background & AimsHBV exhibits wide genetic diversity with at least 9 genotypes (GTs), which differ in terms of prevalence, geographic distribution, natural history, disease progression, and treatment outcome. However, differences in HBV replicative capacity, gene expression, and infective capability across different GTs remain incompletely understood. Herein, we aimed to study these crucial aspects using newly constructed infectious clones covering the major HBV GTs.MethodsThe replicative capacity of infectious clones covering HBV GTs A-E was analyzed in cell lines, primary hepatocytes and humanized mice. Host responses and histopathology induced by the different HBV GTs were characterized in hydrodynamically injected mice. Differences in treatment responses to entecavir and various HBV capsid inhibitors were also quantified across the different genetically defined GTs.ResultsPatient-derived HBV infectious clones replicated robustly both in vitro and in vivo. GTs A and D induce more pronounced intrahepatic and proinflammatory cytokine responses which correlated with faster viral clearance. Notably, all 5 HBV clones robustly produced viral particles following transfection into HepG2 cells, and these particles were infectious in HepG2-NTCP cells, primary human hepatocytes and human chimeric mice. Notably, GT D virus exhibited higher infectivity than GTs A, B, C and E in vitro, although it was comparable to GT A and B in the human liver chimeric mice in vivo. HBV capsid inhibitors were more readily capable of suppressing HBV GTs A, B, D and E than C.ConclusionsThe infectious clones described here have broad utility as genetic tools that can mechanistically dissect intergenotypic differences in antiviral immunity and pathogenesis and aid in HBV drug development and screening.Lay summaryThe hepatitis B virus (HBV) is a major contributor to human morbidity and mortality. HBV can be categorized into a number of genotypes, based on their specific genetic make-up, of which 9 are well known. We isolated and cloned the genomes of 5 of these genotypes and used them to create valuable tools for future research on this clinically important virus.

Biology and molecular pathogenesis of hepatitis B virus infection

Hepatitis B virus (HBV) remains a global public health infection. Over the past several decades, the basic principles of HBV gene expression and replication as well as the viral and host determinants governing infection outcomes have been largely uncovered. The clinical manifestations of HBV infection vary from an acute and chronic form of the disease. During the acute phase of the infection, the disease manifestations vary from subclinical hepatitis to anicteric hepatitis, icteric hepatitis, and fulminant hepatitis while during the chronic infection, manifest in different from ranging from asymptomatic carrier state to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The clinical outcome of the infection depends upon the level of HBV replication, age at infection, and the immune status of the host. HBV integration into the host genome often serves as a relevant source of hepatitis B surface antigen (HBsAg) expression during chronic infection, possibly triggering an immune response. If HBV is not eliminated, a delicate balance between viral replication and immune defence prevails which may lead to chronic hepatitis and liver cirrhosis. It is generally acknowledged that the humoral antibody response contributes to the clearance of circulating virus particles and the prevention of viral spread within the host while the cellular immune response eliminates infected cells.

Efficient silencing of hepatitis B virus S gene through CRISPR-mediated base editing.

Hepatitis B virus (HBV) infection is a major risk factor of liver cirrhosis and hepatocellular carcinoma. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) has been used to precisely edit the HBV genome and eliminate HBV through non‐homologous end‐joining repair of double‐stranded break (DSB). However, the CRISPR/Cas9‐mediated DSB triggers instability of host genome and exhibits low efficiency to edit genome, limiting its application. CRISPR cytidine base editors (CBEs) could silence genes by generating a premature stop codon. Here we developed a CRISPR base editor approach to precisely edit single nucleotide within the HBV genome to impair HBV gene expression. Specifically, a single‐guide RNA (sgRNA) was designed to edit the 30th codon of HBV S gene, which encodes HBV surface antigen (HBsAg), from CAG (glutamine) to stop codon TAG. We next used human hepatoma PLC/PRF/5 cells carrying the HBV genome to establish a cell line that expresses a CBE (PLC/PRF/5‐CBE). Lentivirus was used to introduce sgRNA into PLC/PRF/5‐CBE cells. Phenotypically, 71% of PLC/PRF/5‐CBE cells developed a premature stop codon within the S gene. Levels of HBs messenger RNA were significantly decreased. A 92% reduction of HBsAg secretion was observed in PLC/PRF/5‐CBE cells. The intracellular HBsAg was also reduced by 84% after treatment of gRNA_S. Furthermore, no off‐target effect was detected in predicted off‐target loci within the HBV genome. Sequencing confirmed that 95%, 93%, 93%, 9%, and 72% S gene sequences of HBV genotypes B, C, F, G, and H had the binding site of sgRNA. Conclusion: Our findings indicate that CRISPR‐mediated base editing is an efficient approach to silence the HBV S gene, suggesting its therapeutic potential to eliminate HBV.

Macrophages activated by hepatitis B virus have distinct metabolic profiles and suppress the virus via IL-1β to downregulate PPARα and FOXO3.

SUMMARYMacrophages display phenotypic plasticity and can be induced by hepatitis B virus (HBV) to undergo either M1-like pro-inflammatory or M2-like anti-inflammatory polarization. Here, we report that M1-like macrophages stimulated by HBV exhibit a strong HBV-suppressive effect, which is diminished in M2-like macrophages. Tran-scriptomic analysis reveals that HBV induces the expression of interleukin-1β (IL-1β) in M1-like macrophages, which display a high oxidative phosphorylation (OXPHOS) activity distinct from that of conventional M1-like macrophages. Further analysis indicates that OXPHOS attenuates the expression of IL-1β, which suppresses the expression of peroxisome proliferator-activated receptor α (PPARα) and forkhead box O3 (FOXO3) in hepatocytes to suppress HBV gene expression and replication. Moreover, multiple HBV proteins can induce the expression of IL-1β in macrophages. Our results thus indicate that macrophages can respond to HBV by producing IL-1β to suppress HBV replication. However, HBV can also metabolically reprogram macrophages to enhance OXPHOS to minimize this host antiviral response.

Identification and functional characterization of miR-451a as a novel plasma-based biomarker for occult hepatitis B virus infection

BackgroundNumerous studies have indicated that miRNAs might play significant roles in the development of hepatitis B virus (HBV) infection. while the miRNAs in occult HBV infection (OBI) are still largely unknown. MethodsInitially, 15 HBV infection-related miRNAs in plasma of 10 OBI and 10 healthy controls (HCs) was analyzed by qRT-PCR. Significantly dysregulated miRNAs were subsequently validated in another 64 OBI, 20HCs, 31 chronic hepatitis B (CHB) and 20 asymptomatic HBsAg carriers (ASC). Furthermore, the potential biological functions and molecular mechanisms of miR-451a in HBV infection were investigated using HBV-expressing hepatoma cell lines. ResultsCompared to HCs, plasma miR-451a and miR-340-3p were significantly up-regulated in OBI, ASC and CHB patients, while no significant difference was found among OBI, ASC and CHB patients. ROC curve analysis indicated that both plasma miR-451a and miR-340-3p could moderately distinguish OBI from HCs, with AUCs of 0.76 and 0.78, respectively. When combined, the differentiation efficiency of this miRNA panel was better, with an AUC of 0.82. While, they both could not specifically separate the stage of chronic HBV infection. Functional experiments showed that overexpression of miR-451a might suppress HBV replication and gene expression in hepatoma cell lines. Mechanistically, miR-451a might inhibit HBV replication and gene expression by directly targeting ATF2. ConclusionsA plasma panel, including miR-340-3p and miR-451a that might suppress HBV replication by targeting ATF2, has the potential as biomarkers for HBV infection. In the setting of blood donations, this panel would be more practical to moderately differentiate OBI in HBsAg-negative donors.

Exploring new targets for the treatment of hepatitis-B virus and hepatitis-B virus-associated hepatocellular carcinoma: A new perspective in bioinformatics.

Background:Hepatitis B Virus (HBV) infection is a global public health problem. After infection, patients experience a natural course from chronic hepatitis to cirrhosis and even Hepatitis B associated Hepatocellular Carcinoma (HBV-HCC). With the multi-omics research, many differentially expressed genes from chronic hepatitis to HCC stages have been discovered. All these provide important clues for new biomarkers and therapeutic targets. The purpose of this study is to explore the differential gene expression of HBV and HBV-related liver cancer, and analyze their enrichments and significance of related pathways.Methods:In this study, we downloaded four microarray datasets GSE121248, GSE67764, GSE55092, GSE55092 and GSE83148 from the Gene Expression Omnibus (GEO) database. Using these four datasets, patients with chronic hepatitis B (CHB) differentially expressed genes (CHB DEGs) and patients with HBV-related HCC differentially expressed genes (HBV-HCC DEGs) were identified. Then Protein–protein Interaction (PPI) network analysis, Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to excavate the functional interaction of these two groups of DEGs and the common DEGs. Finally, the Kaplan website was used to analyze the role of these genes in HCC prognostic.Results:A total of 241 CHB DEGs, 276 HBV-HCC DEGs, and 4 common DEGs (cytochrome P450 family 26 subfamily A member 1 (CYP26A1), family with sequence similarity 110 member C(FAM110C), SET and MYND domain containing 3(SMYD3) and zymogen granule protein 16(ZG16)) were identified. CYP26A1, FAM110C, SMYD3 and ZG16 exist in 4 models and interact with 33 genes in the PPI network of CHB and HBV-HCC DEGs,. GO function analysis showed that: CYP26A1, FAM110C, SMYD3, ZG16, and the 33 genes in their models mainly affect the regulation of synaptic vesicle transport, tangential migration from the subventricular zone to the olfactory bulb, cellular response to manganese ion, protein localization to mitochondrion, cellular response to dopamine, negative regulation of neuron death in the biological process of CHB. In the biological process of HBV-HCC, they mainly affect tryptophan catabolic process, ethanol oxidation, drug metabolic process, tryptophan catabolic process to kynurenine, xenobiotic metabolic process, retinoic acid metabolic process, steroid metabolic process, retinoid metabolic process, steroid catabolic process, retinal metabolic process, and rogen metabolic process. The analysis of the 4 common DEGs related to the prognosis of liver cancer showed that: CYP26A1, FAM110C, SMYD3 and ZG16 are closely related to the development of liver cancer and patient survival. Besides, further investigation of the research status of the four genes showed that CYP26A1 and SMYD3 could also affect HBV replication and the prognosis of liver cancer.Conclusion:CYP26A1, FAM110C, SMYD3 and ZG16 are unique genes to differentiate HBV infection and HBV-related HCC, and expected to be novel targets for HBV-related HCC occurrence and prognostic judgement.

Identification of novel hepatitis B virus therapeutic vaccine candidates derived from polymerase protein.

Hepatitis B virus (HBV) infection is a worldwide health problem with high morbidity and mortality rates. The therapeutic vaccine is a promising method of treatment, and HBV polymerase plays a vital role in viral replication. Therefore, a therapeutic vaccine that binds to HBV DNA polymerase may control HBV infection. We predicted and selected epitopes of polymerase using online databases and analysis software. We then performed molecular docking and peptide binding assays to evaluate the binding energies and affinities between polymerase epitopes and the HLA-A0201 molecule. Finally, we induced T cells from the peripheral blood mononuclear cells (PBMCs) of healthy donors using each epitope and quantified the functions of epitope-specific T cells by IFN-γELISPOT assay, T2 cell cytotoxicity assay, HepG2.2.15 cell cytotoxicity assay and HBV gene expression assays. Four epitopes (RVTGGVFLV, GLLGFAAPF, LLDDEAGPL and YMDDVVLGA) had low binding energy and two epitopes (RVTGGVFLV and GLLGFAAPF) had a high binding affinity. The T cells stimulated by two epitopes (GLLGFAAPF and HLYSHPIIL) had a greater ability to induce immune response and suppress HBV. The HBV DNA polymerase epitopes identified in this study are promising targets for designing an epitope-based therapeutic vaccine against HBV.