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1.
PLoS Pathog ; 16(8): e1008802, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822428

RESUMO

Hepatitis B virus (HBV) is a worldwide health problem without curative treatments. Investigation of the regulation of HBV biosynthesis by class I and II histone deacetylases (HDACs) demonstrated that catalytically active HDAC5 upregulates HBV biosynthesis. HDAC5 expression increased both the stability and splicing of the HBV 3.5 kb RNA without altering the translational efficiency of the viral pregenomic or spliced 2.2 kb RNAs. Together, these observations point to a broader role of HDAC5 in regulating RNA splicing and transcript stability while specifically identifying a potentially novel approach toward antiviral HBV therapeutic development.


Assuntos
Genoma Viral , Vírus da Hepatite B/metabolismo , Hepatite B/virologia , Histona Desacetilases/metabolismo , Estabilidade de RNA , RNA Viral/biossíntese , RNA Viral/química , Regulação Viral da Expressão Gênica , Células Hep G2 , Vírus da Hepatite B/genética , Histona Desacetilases/genética , Humanos , Transcrição Genética , Replicação Viral
2.
PLoS One ; 15(6): e0234773, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559248

RESUMO

Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related death worldwide. Patients with hepatitis B virus (HBV) pre-S mutants in liver tissues or blood have been regarded as a high-risk population for HCC development and recurrence. Detection of pre-S mutants in clinical specimens is thus important for early diagnosis and prognosis of HCC to improve patient survival. Recently, we have developed a next-generation sequencing (NGS)-based platform that can quantitatively detect pre-S mutants in patient plasma with superior sensitivity and accuracy. In this study, we compared the pre-S genotyping results from plasma by the NGS-based analysis with those from liver tissues by the immunohistochemistry (IHC)-based analysis in 30 HBV-related HCC patients. We demonstrated that the detection rate of pre-S mutants was significantly higher by NGS- than by IHC-based analysis. There was a moderate to good agreement between both analyses in detection of pre-S mutants. Compared with the IHC, the NGS-based detection of pre-S mutants in patient plasma could determine the patterns of pre-S mutants in liver tissues more efficiently in a noninvasive manner. Our data suggest that the NGS-based platform may represent a promising approach for detection of pre-S mutants as biomarkers of HBV-related HCC in clinical practice.


Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Fígado/virologia , Precursores de Proteínas/genética , Adulto , Idoso , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , DNA Viral/sangue , DNA Viral/metabolismo , Feminino , Genótipo , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/complicações , Hepatite B Crônica/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Fígado/patologia , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Mutação , Precursores de Proteínas/sangue , Análise de Sequência de DNA
4.
PLoS One ; 15(5): e0233702, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32442221

RESUMO

Liver fibrosis is a manifestation of chronic liver injury. It leads to hepatic dysfunction and is a critical element in the pathogenesis of cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cells (HSC) plays a central role in liver fibrogenesis of different etiologies. To elucidate the molecular mechanism of this phenomenon, it is important to analyze the changes in gene expression that accompany the HSC activation process. In this study, we isolated quiescent and activated HSCs from control mice and mice with CCl4-induced liver fibrosis, respectively, and performed RNA sequencing to compare the differences in gene expression patterns between the two types of HSCs. We also reanalyzed public gene expression data for fibrotic liver tissues isolated from patients with HBV infection, HCV infection, and nonalcoholic fatty liver disease to investigate the gene expression changes during liver fibrosis of these three etiologies. We detected 146 upregulated and 18 downregulated genes in activated HSCs, which were implicated in liver fibrosis as well. Among the overlapping genes, seven transcription factor-encoding genes, ARID5B, GATA6, MITF, PBX1, PLAGL1, SOX4, and SOX9, were upregulated, while one, RXRA, was downregulated. These genes were suggested to play a critical role in HSC activation, and subsequently, in the promotion of liver fibrosis. We undertook the RNA sequencing of quiescent and activated HSCs and analyzed the expression profiles of genes associated with HSC activation in liver fibrotic tissues from different liver diseases, and also aimed to elucidate the changes in gene expression patterns associated with HSC activation and liver fibrosis.


Assuntos
Hepacivirus/metabolismo , Células Estreladas do Fígado/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Hepatite C/metabolismo , Cirrose Hepática/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Células Estreladas do Fígado/patologia , Células Estreladas do Fígado/virologia , Hepatite B/patologia , Hepatite C/patologia , Humanos , Cirrose Hepática/patologia , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , Fatores de Transcrição/biossíntese
5.
PLoS Pathog ; 16(3): e1008459, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226051

RESUMO

Hepatitis B virus (HBV) delivers a partially double-stranded, relaxed circular (RC) DNA genome in complete virions to the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, which establishes and sustains viral infection. An overlength pregenomic RNA (pgRNA) is then transcribed from CCC DNA and packaged into immature nucleocapsids (NCs) by the viral core (HBc) protein. pgRNA is reverse transcribed to produce RC DNA in mature NCs, which are then enveloped and secreted as complete virions, or delivered to the nucleus to replenish the nuclear CCC DNA pool. RC DNA, whether originating from extracellular virions or intracellular mature NCs, must be released upon NC disassembly (uncoating) for CCC DNA formation. HBc is known to undergo dynamic phosphorylation and dephosphorylation at its C-terminal domain (CTD) to facilitate pgRNA packaging and reverse transcription. Here, two putative phosphorylation sites in the HBc N-terminal domain (NTD), S44 and S49, were targeted for genetic and biochemical analysis to assess their potential roles in viral replication. The NTD mutant that mimics the non-phosphorylated state (N2A) was competent in all steps of viral replication tested from capsid assembly, pgRNA packaging, reverse transcription, to virion secretion, except for a decrease in CCC DNA formation. On the other hand, the phosphor-mimetic mutant N2E showed a defect in the early step of pgRNA packaging but enhanced the late step of mature NC uncoating and consequently, increased CCC DNA formation. N2E also enhanced phosphorylation in CTD and possibly elsewhere in HBc. Furthermore, inhibition of the cyclin-dependent kinase 2 (CDK2), which is packaged into viral capsids, could block CCC DNA formation. These results prompted us to propose a model whereby rephosphorylation of HBc at both NTD and CTD by the packaged CDK2, following CTD dephosphorylation during NC maturation, facilitates uncoating and CCC DNA formation by destabilizing mature NCs.


Assuntos
DNA Circular/metabolismo , DNA Viral/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Modelos Biológicos , Nucleocapsídeo/metabolismo , Desenvelopamento do Vírus , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , DNA Circular/genética , DNA Viral/genética , Células HEK293 , Células Hep G2 , Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Nucleocapsídeo/genética , Fosforilação , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo
6.
PLoS One ; 15(4): e0232208, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320459

RESUMO

INTRODUCTION: Hepatitis B virus infection is a global public health problem. Though, the disease is endemic in sub-Saharan Africa, little is known about its epidemiology among pregnant women in Ghana. This study sought to determine the seroprevalence of Hepatitis B virus infection and associated factors among pregnant women attending antenatal care at Korle-Bu Teaching Hospital; Ghana's largest hospital. METHODS: We conducted a facility-based cross-sectional survey among 232 antenatal attendants. Participants were recruited using systematic random sampling technique and screened with HBsAg Rapid Test. Data was analyzed with the aid of Statistical Package for Social Sciences (SPSS), version 23.0. Results were presented using descriptive statistics, Fisher's Exact test and Logistic Regression analysis. RESULTS: Two hundred and twenty-one (221) of the total sample (n = 232) agreed to participate in this study; representing a response rate of 95%. The mean age of the participants was 31 years and standard deviation of 5.3. The mean gestational period at recruitment was 28 weeks and standard deviation of 6.8. Majority of the participants were married (83.3%), parous (69.6%), educated (91.4%) and employed (90.5%). The prevalence of HBsAg was 7.7%. We found no significant association between socio-demographic characteristics of the participants and HBV infection. CONCLUSION: Seroprevalence of 7.7% indicates moderate endemicity. Socio-demographic characteristics did not influence HBV infection among pregnant women attending antenatal care at Korle-Bu Teaching Hospital. The findings provide empirical evidence that will contribute to knowledge of HBV epidemiology in Ghana.


Assuntos
Vírus da Hepatite B/patogenicidade , Hepatite B/epidemiologia , Adolescente , Adulto , Estudos Transversais , Feminino , Gana/epidemiologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hospitais de Ensino/métodos , Humanos , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Cuidado Pré-Natal/métodos , Prevalência , Fatores de Risco , Estudos Soroepidemiológicos , Adulto Jovem
7.
Int J Clin Oncol ; 25(7): 1327-1333, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32200482

RESUMO

BACKGROUND: Patients with hepatitis B virus (HBV) infection have a risk of reactivation after chemotherapy. All patients undergoing chemotherapy should be screened for HBV infection. No large-scale studies have been conducted to examine HBV screening practice in Japan. METHODS: We analyzed health insurance claims equivalent data linked with a nationwide hospital-based cancer registry. Patients diagnosed with cancer in 2014, who were aged 20 years and older and those who underwent systemic anticancer treatment in 2014-15 were included. We assessed the HBV screening rates by the HBsAg or anti-HBc tests, HBV-DNA tests, and entecavir prescriptions. Multiple logistic regression models were used to identify factors related to the receipt of screening. RESULTS: Of 177,597 patients (mean [SD] age, 65.6 [12.2] years), 82.6% and 12.9% patients had a solid tumor and hematologic malignancy, respectively. Among them, 88.1%, 6.3%, and 5.5% received cytotoxic chemotherapy, targeted therapy, and anti-CD20 antibodies, respectively. Overall, 70.6% of patients were screened. The positive predictor of HBV screening was receiving anti-CD20 antibodies [odds ratio (OR); 2.23, 95% confidence interval (CI) 2.06-2.41, p < 0.001] and negative predictors were age ≥ 85 (OR 0.76, 95% CI 0.71-0.81), age 75-84 (OR 0.77, 95% CI 0.75-0.79) and targeted therapy (OR 0.69, 95% CI 0.67-0.72). Among the screened patients, 13.2% were tested for HBV-DNA, and 1.49% were prescribed entecavir. CONCLUSIONS: The HBV screening rate in Japan is higher than in other countries. Further improvement of the HBV screening rate is needed to prevent reactivation and avoidable deaths of patients with HBV infection.


Assuntos
Vírus da Hepatite B/metabolismo , Hepatite B/diagnóstico , Neoplasias/virologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/uso terapêutico , Antígenos CD20/imunologia , Antineoplásicos/uso terapêutico , Feminino , Guanina/análogos & derivados , Guanina/uso terapêutico , Hepatite B/epidemiologia , Hepatite B/etiologia , Hepatite B/prevenção & controle , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Humanos , Japão/epidemiologia , Masculino , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Razão de Chances , Ativação Viral/efeitos dos fármacos
8.
PLoS Pathog ; 16(2): e1008338, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32059034

RESUMO

Interferon (IFN) stimulates a whole repertoire of cellular genes, collectively referred to as ISGs (Interferon-stimulated genes). ISG20, a 3´-5´ exonuclease enzyme, has been previously shown to bind and degrade hepatitis B Virus (HBV) transcripts. Here, we show that the N6-methyladenosine (m6A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m6A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m6A site within HBV transcripts and confirmed that methylation at nucleotide A1907 regulates HBV lifecycle. In this report, we now show that the methylation at A1907 is a critical regulator of IFN-α mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m6A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20D94G, we further demonstrated that ISG20 forms a complex with m6A modified HBV RNA and YTHDF2 protein. Due to terminal redundancy, HBV genomic nucleotide A1907 position is acquired twice by pregenomic RNA (pgRNA) during transcription and therefore the sites of methylation are encoded within 5´ and 3´ epsilon stem loops. We generated HBV mutants that lack m6A site at either one (5´ or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m6A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m6A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m6A modification of RNA in IFN-α induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-α mediated viral RNA degradation.


Assuntos
Exorribonucleases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Antivirais/farmacologia , Exonucleases/metabolismo , Exorribonucleases/genética , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Interferon-alfa/farmacologia , Interferons/metabolismo , Metiltransferases/metabolismo , Estabilidade de RNA/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Replicação Viral/fisiologia
9.
Am J Physiol Gastrointest Liver Physiol ; 318(3): G401-G409, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31905024

RESUMO

Hepatitis B virus (HBV)-related hepatocarcinogenesis is not necessarily associated with the liver fibrotic stage and is occasionally seen at early fibrotic stages. MicroRNAs (miRNAs) are essentially 18- to 22-nucleotide-long endogenous noncoding RNAs. Aberrant miRNA expression is a common feature of various human cancers. The aberrant expression of specific miRNAs has been shown in hepatocellular carcinoma (HCC) tissue compared with nontumor tissue. Thus, we examined targetable miRNAs as a potential new biomarker related to the high risk of HBV-related hepatocarcinogenesis, toward the prevention of cancer-related deaths. HCC tissue samples from 29 patients who underwent hepatectomy at our hospital in 2002-2013 were obtained. We extracted the total RNA and analyzed it by microRNA array, real-time RT-PCR, and three comparisons: 1) HBV-related HCC and adjacent nontumor tissue, 2) HCV-related HCC and adjacent nontumor tissue, and 3) non-HBV-, non-HCV-related HCC and adjacent nontumor tissue. We also performed a functional analysis of miRNAs specific for HBV-related HCC by using HBV-positive HCC cell lines. MiR-210-3p expression was significantly increased only in the HBV-related HCC tissue samples. MiR-210-3p expression was upregulated, and the levels of its target genes were reduced in the HBV-positive HCC cells. The inhibition of miR-210-3p enhanced its target gene expression in the HBV-positive HCC cells. In addition, miR-210-3p regulated the HBx expression in HBV-infected Huh7/NTCP cells. The enhanced expression of miR-210-3p was detected specifically in HBV-related HCC and regulated various target genes, including HBx in the HBV-positive HCC cells. MiR-210-3p might, thus, be a new biomarker for the risk of HBV-related HCC.NEW & NOTEWORTHY Our present study demonstrated that miR-210-3p is the only microRNA with enhanced expression in HBV-related HCC, and the enhanced expression of miR-210-3p upregulates HBx expression. Therefore, miR-210-3p might be a pivotal biomarker of HBV-related hepatocarcinogenesis, and the inhibition of miR-210-3p could prevent inducing hepatocarcinogenesis related to HBV infection.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transformação Celular Viral , Vírus da Hepatite B/metabolismo , Hepatite B/virologia , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Transativadores/metabolismo , Idoso , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Hepatite B/complicações , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Masculino , MicroRNAs/genética , Transdução de Sinais , Transativadores/genética , Replicação Viral
10.
Am J Physiol Gastrointest Liver Physiol ; 318(1): G162-G173, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31604033

RESUMO

Hepatitis B virus (HBV) exploits multiple strategies to evade host immune surveillance. Programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) signaling plays a critical role in regulating T cell homeostasis. However, it remains largely unknown as to how HBV infection elevates PD-L1 expression in hepatocytes. A mouse model of HBV infection was established by hydrodynamic injection with a vector containing 1.3-fold overlength HBV genome (pHBV1.3) via the tail vein. Coculture experiments with HBV-expressing hepatoma cells and Jurkat T cells were established in vitro. We observed significant decrease in the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and increase in ß-catenin/PD-L1 expression in liver tissues from patients with chronic hepatitis B and mice subjected to pHBV1.3 hydrodynamic injection. Mechanistically, decrease in PTEN enhanced ß-catenin/c-Myc signaling and PD-L1 expression in HBV-expressing hepatoma cells, which in turn augmented PD-1 expression, lowered IL-2 secretion, and induced T cell apoptosis. However, ß-catenin disruption inhibited PTEN-mediated PD-L1 expression, which was accompanied by decreased PD-1 expression, and increased IL-2 production in T cells. Luciferase reporter assays revealed that c-Myc stimulated transcriptional activity of PD-L1. In addition, HBV X protein (HBx) and HBV polymerase (HBp) contributed to PTEN downregulation and ß-catenin/PD-L1 upregulation. Strikingly, PTEN overexpression in hepatocytes inhibited ß-catenin/PD-L1 signaling and promoted HBV clearance in vivo. Our findings suggest that HBV-triggered PTEN/ß-catenin/c-Myc signaling via HBx and HBp enhances PD-L1 expression, leading to inhibition of T cell response, and promotes HBV immune evasion.NEW & NOTEWORTHY This study demonstrates that during HBV infection, HBV can increase PD-L1 expression via PTEN/ß-catenin/c-Myc signaling pathway, which in turn inhibits T cell response and ultimately promotes HBV immune evasion. Targeting this signaling pathway is a potential strategy for immunotherapy of chronic hepatitis B.


Assuntos
Antígeno B7-H1/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/metabolismo , Hepatócitos/enzimologia , Evasão da Resposta Imune , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linfócitos T/enzimologia , beta Catenina/metabolismo , Animais , Técnicas de Cocultura , Modelos Animais de Doenças , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Células Jurkat , Ativação Linfocitária , Masculino , Camundongos Endogâmicos BALB C , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/virologia , Transativadores/genética , Transativadores/metabolismo
11.
Virology ; 539: 92-103, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31706164

RESUMO

Hepatitis B virus (HBV) X protein (HBx) has been reported to counteract the innate immune responses through interfering with the pattern recognition receptors signaling activated by retinoic acid-inducible gene-I (RIG-I)-mitochondrial antiviral signaling protein (MAVS). Here, we showed that, compared to the HBx derived from genotype (gt) A, C and D, HBx of gtB exhibited more potent inhibitory activity on the RIG-I-MAVS-mediated interferon-ß promoter activation. Functional analysis of the genotype-associated differences in amino acid sequence and the reciprocal mutation experiments in transient-transfection and infection cell models revealed that HBx with asparagine (N) and glutamic acid (E) at 118-119 positions inhibited RIG-I signaling and interacted with MAVS more efficiently than that with lysine (K) and aspartic acid (D). An impaired RIG-I-induced MAVS aggregation was observed in the presence of HBx-118N119E while MAVS-TRAF3 interaction was not affected. These results implicated that HBx gene heterogeneity may affect the innate immune responses to HBV infection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína DEAD-box 58/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B/metabolismo , Transativadores/metabolismo , Aminoácidos , Células Cultivadas , Genótipo , Células HEK293 , Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Imunidade Inata , Interferon beta/genética , Interferon beta/metabolismo , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Transativadores/química , Transativadores/genética
12.
Int J Mol Sci ; 20(17)2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31480501

RESUMO

Hepatitis B virus (HBV) infection is a major health problem affecting about 300 million people globally. Although successful administration of a prophylactic vaccine has reduced new infections, a cure for chronic hepatitis B (CHB) is still unavailable. Current anti-HBV therapies slow down disease progression but are not curative as they cannot eliminate or permanently silence HBV covalently closed circular DNA (cccDNA). The cccDNA minichromosome persists in the nuclei of infected hepatocytes where it forms the template for all viral transcription. Interactions between host factors and cccDNA are crucial for its formation, stability, and transcriptional activity. Here, we summarize the reported interactions between HBV cccDNA and various host factors and their implications on HBV replication. While the virus hijacks certain cellular processes to complete its life cycle, there are also host factors that restrict HBV infection. Therefore, we review both positive and negative regulation of HBV cccDNA by host factors and the use of small molecule drugs or sequence-specific nucleases to target these interactions or cccDNA directly. We also discuss several reporter-based surrogate systems that mimic cccDNA biology which can be used for drug library screening of cccDNA-targeting compounds as well as identification of cccDNA-related targets.


Assuntos
DNA Circular/metabolismo , Vírus da Hepatite B/genética , Hepatite B/genética , Hepatócitos/virologia , Replicação Viral , Animais , DNA Viral , Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Hepatócitos/metabolismo , Humanos
13.
Inflamm Res ; 68(12): 1025-1034, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31531682

RESUMO

OBJECTIVE: Saikosaponin c (SSc), a compound purified from the traditional Chinese herb of Radix Bupleuri was previously identified to exhibit anti-HBV replication activity. However, the mechanism through which SSc acts against HBV remains unknown. In this study, we investigated the mechanism of SSc mediated anti-HBV activity. METHODS: HepG2.2.15 cells were cultured at 37 â„ƒ in the presence of 1-40 µg/mL of SSc or DMSO as a control. The expression profile of HBV markers, cytokines, HNF1α and HNF4α were investigated by real-time quantitative PCR, Elisa, Western blot and Dot blotting. Knockdown of HNF1α or HNF4α in HepG2.2.15 cells was mediated by two small siRNAs specifically targeting HNF1α or HNF4α. RESULTS: We found that SSc stimulates IL-6 expression, leading to attenuated HNF1α and HNF4α expression, which further mediates suppression of HBV pgRNA synthesis. Knockdown of HNF1α or HNF4α in HepG2.2.15 cells by RNA interference abrogates SSc's anti-HBV role. Moreover, SSc is effective to both wild-type and drug-resistant HBV mutants. CONCLUSION: SSc inhibits pgRNA synthesis by targeting HNF1α and HNF4α. These results indicate that SSc acts as a promising compound for modulating pgRNA transcription in the therapeutic strategies against HBV infection.


Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Ácido Oleanólico/análogos & derivados , RNA Viral/biossíntese , RNA/biossíntese , Saponinas/farmacologia , Células Hep G2 , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Humanos , Ácido Oleanólico/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética
14.
Int J Nanomedicine ; 14: 6601-6613, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496701

RESUMO

Purpose: The primary goal of the present study was to explore and evaluate the highly conserved Neisserial surface protein A (NspA) molecule, fused with truncated HBV virus-like particles (VLPs), as a candidate vaccine against the virulent Neisseria meningitidis serogroup B (NMB). Methods: NspA was inserted into the major immunodominant region of the truncated hepatitis B virus core protein (HBc; amino acids 1-144). The chimeric protein, HBc-N144-NspA, was expressed from a prokaryotic vector and generated HBc-like particles, as determined by transmission electron microscopy. Further, the chimeric protein and control proteins were used to immunize mice and the resulting immune responses evaluated by flow cytometry, enzyme-linked immunosorbent assay, and analysis of serum bactericidal activity (SBA) titer. Results: Evaluation of the immunogenicity of the recombinant HBc-N144-NspA protein showed that it elicited the production of high levels of NspA-specific total IgG. The SBA titer of HBc-N144-NspA/F reached 1:16 2 weeks after the last immunization in BALB/c mice, when human serum complement was included in the vaccine. Immunization of HBc-N144-NspA, even without adjuvant, induced high levels of IL-4 and a high IgG1 to IgG2a ratio, confirming induction of an intense Th2 immune response. Levels of IL-17A increased rapidly in mice after the first immunization with HBc-N144-NspA, indicating the potential for this vaccine to induce a mucosal immune response. Meanwhile, the immunization of HBc-N144-NspA without adjuvant induced only mild inflammatory infiltration into the mouse muscle tissue. Conclusion: This study demonstrates that modification using HBc renders NspA a candidate vaccine, which can trigger protective immunity against NMB.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vírus da Hepatite B/metabolismo , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis/patogenicidade , Sorogrupo , Vírion/metabolismo , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Citocinas/metabolismo , Escherichia coli/metabolismo , Feminino , Imunidade , Imunização , Inflamação/patologia , Ativação Linfocitária/imunologia , Infecções Meningocócicas/patologia , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Teste Bactericida do Soro , Baço/microbiologia , Linfócitos T/imunologia , Vacinação , Virulência
15.
J Biol Chem ; 294(38): 14043-14054, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31366735

RESUMO

Adenosine deaminases acting on RNA-1 (ADAR1) involves adenosine to inosine RNA editing and microRNA processing. ADAR1 is known to be involved in the replication of various viruses, including hepatitis C and D. However, the role of ADAR1 in hepatitis B virus (HBV) infection has not yet been elucidated. Here, for the first time, we demonstrated ADAR1 antiviral activity against HBV. ADAR1 has two splicing isoforms in human hepatocytes: constitutive p110 protein and interferon-α (IFN-α)-responsive p150 protein. We found that overexpression of ADAR1 decreased HBV RNA in an HBV culture model. A catalytic-site mutant ADAR1 also decreased HBV RNA levels, whereas another adenosine deaminases that act on the RNA (ADAR) family protein, ADAR2, did not. Moreover, the induction of ADAR1 by stimulation with IFN-α also reduced HBV RNA levels. Decreases in endogenous ADAR1 expression by knock-down or knock-out increased HBV RNA levels. A major hepatocyte-specific microRNA, miRNA-122, was found to be positively correlated with ADAR1 expression, and exogenous miRNA-122 decreased both HBV RNA and DNA, whereas, conversely, transfection with a miRNA-122 inhibitor increased them. The reduction of HBV RNA by ADAR1 expression was abrogated by p53 knock-down, suggesting the involvement of p53 in the ADAR1-mediated reduction of HBV RNA. This study demonstrated, for the first time, that ADAR1 plays an antiviral role against HBV infection by increasing the level of miRNA-122 in hepatocytes.


Assuntos
Adenosina Desaminase/metabolismo , Vírus da Hepatite B/fisiologia , MicroRNAs/genética , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/fisiologia , Adenosina Desaminase/genética , Linhagem Celular , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , MicroRNAs/metabolismo , Isoformas de Proteínas , Edição de RNA , Processamento de RNA , Proteínas de Ligação a RNA/genética
16.
Proc Natl Acad Sci U S A ; 116(34): 17007-17012, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31371507

RESUMO

Shrews, insectivorous small mammals, pertain to an ancient mammalian order. We screened 693 European and African shrews for hepatitis B virus (HBV) homologs to elucidate the enigmatic genealogy of HBV. Shrews host HBVs at low prevalence (2.5%) across a broad geographic and host range. The phylogenetically divergent shrew HBVs comprise separate species termed crowned shrew HBV (CSHBV) and musk shrew HBV (MSHBV), each containing distinct genotypes. Recombination events across host orders, evolutionary reconstructions, and antigenic divergence of shrew HBVs corroborated ancient origins of mammalian HBVs dating back about 80 million years. Resurrected CSHBV replicated in human hepatoma cells, but human- and tupaia-derived primary hepatocytes were resistant to hepatitis D viruses pseudotyped with CSHBV surface proteins. Functional characterization of the shrew sodium taurocholate cotransporting polypeptide (Ntcp), CSHBV/MSHBV surface peptide binding patterns, and infection experiments revealed lack of Ntcp-mediated entry of shrew HBV. Contrastingly, HBV entry was enabled by the shrew Ntcp. Shrew HBVs universally showed mutations in their genomic preCore domains impeding hepatitis B e antigen (HBeAg) production and resembling those observed in HBeAg-negative human HBV. Deep sequencing and in situ hybridization suggest that HBeAg-negative shrew HBVs cause intense hepatotropic monoinfections and low within-host genomic heterogeneity. Geographical clustering and low MSHBV/CSHBV-specific seroprevalence suggest focal transmission and high virulence of shrew HBVs. HBeAg negativity is thus an ancient HBV infection pattern, whereas Ntcp usage for entry is not evolutionarily conserved. Shrew infection models relying on CSHBV/MSHBV revertants and human HBV will allow comparative assessments of HBeAg-mediated HBV pathogenesis, entry, and species barriers.


Assuntos
Evolução Molecular , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Modelos Genéticos , Filogenia , Musaranhos/virologia , Proteínas do Envelope Viral/genética , Fatores de Virulência/genética , Animais , Linhagem Celular Tumoral , Hepatite B/genética , Hepatite B/metabolismo , Hepatite B/veterinária , Vírus da Hepatite B/metabolismo , Humanos
17.
Nat Commun ; 10(1): 3192, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324803

RESUMO

Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 Å away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients.


Assuntos
Proteínas Reguladoras de Apoptose/química , Vírus da Hepatite B/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-bcl-2/química , Transativadores/química , Transativadores/fisiologia , Proteína bcl-X/química , Animais , Cristalografia por Raios X , Modelos Animais de Doenças , Células Hep G2 , Vírus da Hepatite B/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutação , Ligação Proteica , Transativadores/genética , Replicação Viral/fisiologia
18.
BMC Cancer ; 19(1): 707, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319796

RESUMO

BACKGROUND: Hepatitis B virus (HBV) is the leading cause of liver cancer, but the mechanisms by which HBV causes liver cancer are poorly understood and chemotherapeutic strategies to cure liver cancer are not available. A better understanding of how HBV requisitions cellular components in the liver will identify novel therapeutic targets for HBV associated hepatocellular carcinoma (HCC). MAIN BODY: The development of HCC involves deregulation in several cellular signalling pathways including Wnt/FZD/ß-catenin, PI3K/Akt/mTOR, IRS1/IGF, and Ras/Raf/MAPK. HBV is known to dysregulate several hepatocyte pathways and cell cycle regulation resulting in HCC development. A number of these HBV induced changes are also mediated through the Wnt/FZD/ß-catenin pathway. The lack of a suitable human liver model for the study of HBV has hampered research into understanding pathogenesis of HBV. Primary human hepatocytes provide one option; however, these cells are prone to losing their hepatic functionality and their ability to support HBV replication. Another approach involves induced-pluripotent stem (iPS) cell-derived hepatocytes. However, iPS technology relies on retroviruses or lentiviruses for effective gene delivery and pose the risk of activating a range of oncogenes. Liver organoids developed from patient-derived liver tissues provide a significant advance in HCC research. Liver organoids retain the characteristics of their original tissue, undergo unlimited expansion, can be differentiated into mature hepatocytes and are susceptible to natural infection with HBV. CONCLUSION: By utilizing new ex vivo techniques like liver organoids it will become possible to develop improved and personalized therapeutic approaches that will improve HCC outcomes and potentially lead to a cure for HBV.


Assuntos
Carcinogênese/metabolismo , Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Neoplasias Hepáticas/virologia , Animais , Apoptose , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Hepatite B/metabolismo , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas , Neoplasias Hepáticas/patologia , Organoides , Medicina de Precisão , Transdução de Sinais
19.
Int J Med Sci ; 16(5): 720-728, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31217740

RESUMO

Objective: This study aims to clarify the changes and clinical significance of red cell distribution width (RDW) during HBV-related chronic diseases, including inactive hepatitis B virus (HBV) carriers, HBV immune tolerant individuals, chronic hepatitis B (CHB) patients and HBV-related hepatocirrhosis patients. Methods: RDW was measured 288 CHB patients, 100 patients with hepatitis B e antigen(HBeAg)-negative chronic HBV infection (inactive carriers), 92 patients with HBeAg-positive chronic HBV infection (immune tolerant), and 272 patients with HBV-related hepatocirrhosis. Their RDW changes were compared with 160 healthy controls. Correlations between RDW and clinical indicators were conducted. For HBeAg+ CHB patients, RDW was measured before and after antiviral therapy. The efficiency of RDW to distinguish hepatocirrhosis from CHB and/or inactive carriers was evaluated by receiver operating characteristic (ROC) curves. Results: RDW was higher in hepatocirrhosis patients than other groups of patients and healthy controls. Besides, HBeAg+ CHB patients possessed higher RDW than HBeAg- CHB patients. For HBeAg+ patients that underwent HBeAg seroconversion after antiviral therapy, RDW was decreased. RDW was positively correlated with total bilirubin and Child-Pugh scores and negatively correlated with albumin among hepatocirrhosis patients. The areas under the curve (AUC) of ROC curves to distinguish hepatocirrhosis from CHB patients was 0.7040 for RDW-standard deviation (RDW-SD) and 0.6650 for RDW-coefficient of variation (RDW-CV), and AUC to distinguish hepatocirrhosis from inactive carriers was 0.7805 for RDW-SD and 0.7991 for RDW-CV. Conclusions: RDW is significantly increased in HBeAg+ CHB patients and patients with HBV-related hepatocirrhosis and could reflect their severity. RDW could help to distinguish hepatocirrhosis from CHB patients and inactive HBV carriers.


Assuntos
Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/sangue , Cirrose Hepática/sangue , Adulto , Diagnóstico Diferencial , Índices de Eritrócitos/imunologia , Feminino , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/imunologia , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Humanos , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Masculino , Pessoa de Meia-Idade
20.
Nat Commun ; 10(1): 2265, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118422

RESUMO

Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC50: 1.4 nM) and interferon-α (IC50: 28 IU/ml, but max. 60-80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC50: 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.


Assuntos
Antivirais/farmacologia , Vírus Delta da Hepatite/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Células Hep G2 , Vírus da Hepatite B/metabolismo , Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Concentração Inibidora 50
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