RESUMO
Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two overlapping reading frames, and during evolution, multiple selective pressures can act on shared nucleotides. This study combines an RNA-based HBV cell culture system with deep mutational scanning (DMS) to uncouple cis- and trans-acting sequence requirements in the HBV genome. The results support a leaky ribosome scanning model for polymerase translation, provide a fitness map of the HBV polymerase at single-nucleotide resolution, and identify conserved prolines adjacent to the HBV polymerase termination codon that stall ribosomes. Further experiments indicated that stalled ribosomes tether the nascent polymerase to its template RNA, ensuring cis-preferential RNA packaging and reverse transcription of the HBV genome.
Assuntos
Vírus da Hepatite B , Transcrição Reversa , Humanos , Genoma Viral/genética , Vírus da Hepatite B/genética , Mutação , Ribossomos/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Linhagem CelularRESUMO
Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.
Assuntos
Linfócitos T CD8-Positivos , Vírus da Hepatite B , Hepatite B Crônica , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Hepatite B Crônica/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Animais , Receptores OX40/metabolismo , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Antígenos CD/metabolismoRESUMO
Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8+ T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-specific naive CD8+ T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administration, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Interleucina-2/imunologia , Células de Kupffer/imunologia , Animais , Hepatite B/imunologia , Tolerância Imunológica/imunologia , Camundongos , Camundongos TransgênicosRESUMO
Hepatitis B virus (HBV) infection affects 240 million people worldwide. A liver-specific bile acid transporter named the sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the cellular receptor for HBV and its satellite, the hepatitis D virus (HDV). NTCP likely acts as a major determinant for the liver tropism and species specificity of HBV and HDV at the entry level. NTCP-mediated HBV entry interferes with bile acid transport in cell cultures and has been linked with alterations in bile acid and cholesterol metabolism in vivo. The human liver carcinoma cell line HepG2, complemented with NTCP, now provides a valuable platform for studying the basic biology of the viruses and developing treatments for HBV infection. This review summarizes critical findings regarding NTCP's role as a viral receptor for HBV and HDV and discusses important questions that remain unanswered.
Assuntos
Vírus da Hepatite B/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proteínas de Transporte/metabolismo , Vírus Delta da Hepatite/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismoRESUMO
Evolutionary changes in the hepatitis B virus (HBV) genome could reflect its adaptation to host-induced selective pressure. Leveraging paired human exome and ultra-deep HBV genome-sequencing data from 567 affected individuals with chronic hepatitis B, we comprehensively searched for the signatures of this evolutionary process by conducting "genome-to-genome" association tests between all human genetic variants and viral mutations. We identified significant associations between an East Asian-specific missense variant in the gene encoding the HBV entry receptor NTCP (rs2296651, NTCP S267F) and mutations within the receptor-binding region of HBV preS1. Through in silico modeling and in vitro preS1-NTCP binding assays, we observed that the associated HBV mutations are in proximity to the NTCP variant when bound and together partially increase binding affinity to NTCP S267F. Furthermore, we identified significant associations between HLA-A variation and viral mutations in HLA-A-restricted T cell epitopes. We used in silico binding prediction tools to evaluate the impact of the associated HBV mutations on HLA presentation and observed that mutations that result in weaker binding affinities to their cognate HLA alleles were enriched. Overall, our results suggest the emergence of HBV escape mutations that might alter the interaction between HBV PreS1 and its cellular receptor NTCP during viral entry into hepatocytes and confirm the role of HLA class I restriction in inducing HBV epitope variations.
Assuntos
Vírus da Hepatite B , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio , Simportadores , Humanos , Vírus da Hepatite B/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/genética , Simportadores/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Hepatite B Crônica/virologia , Hepatite B Crônica/genética , Genoma Viral , Antígenos de Superfície da Hepatite B/genética , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Genômica/métodos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismoRESUMO
Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.
Assuntos
Citidina , Vírus da Hepatite B , RNA Viral , Transcrição Reversa , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Citidina/análogos & derivados , Citidina/metabolismo , Citidina/genética , Humanos , Transcrição Reversa/genética , Metilação , Replicação Viral/genética , Epigênese Genética , Vírion/metabolismo , Vírion/genética , TranscriptomaRESUMO
Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.
Assuntos
Hepatócitos , Fígado , Organoides , Hepatócitos/metabolismo , Hepatócitos/citologia , Organoides/metabolismo , Organoides/citologia , Humanos , Animais , Camundongos , Fígado/metabolismo , Fígado/citologia , Camundongos Endogâmicos C57BL , Diferenciação CelularRESUMO
Mammalian cells have evolved strategies to regulate gene expression when oxygen is limited. Hypoxia-inducible factors (HIF) are the major transcriptional regulators of host gene expression. We previously reported that HIFs bind and activate hepatitis B virus (HBV) DNA transcription under low oxygen conditions; however, the global cellular response to low oxygen is mediated by a family of oxygenases that work in concert with HIFs. Recent studies have identified a role for chromatin modifiers in sensing cellular oxygen and orchestrating transcriptional responses, but their role in the HBV life cycle is as yet undefined. We demonstrated that histone lysine demethylase 4 (KDM4) can restrict HBV, and pharmacological or oxygen-mediated inhibition of the demethylase increases viral RNAs derived from both episomal and integrated copies of the viral genome. Sequencing studies demonstrated that KDM4 is a major regulator of the hepatic transcriptome, which defines hepatocellular permissivity to HBV infection. We propose a model where HBV exploits cellular oxygen sensors to replicate and persist in the liver. Understanding oxygen-dependent pathways that regulate HBV infection will facilitate the development of physiologically relevant cell-based models that support efficient HBV replication.
Assuntos
Vírus da Hepatite B , Histona Desmetilases com o Domínio Jumonji , Oxigênio , Replicação Viral , Humanos , DNA Viral/genética , Genoma Viral/genética , Hepatite B/enzimologia , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Fígado/virologia , Oxigênio/metabolismo , Plasmídeos/genética , Transcriptoma , Replicação Viral/genéticaRESUMO
Studies have traditionally focused on the role of T cells in chronic hepatitis B (CHB), but recent evidence supports a role for B cells. The enrichment of so-called atypical memory (AtM) B cells, which show reduced signaling and impaired differentiation, is believed to be a characteristic feature of CHB, potentially contributing to the observed dysfunctional anti-HBsAg B-cell responses. Our study, involving 62 CHB patients across clinical phases, identified AtM B cells expressing IFNLR1 and interferon-stimulated genes. Contrary to previous reports, we found relatively low frequencies of AtM B cells in the liver, comparable to peripheral blood. However, liver plasma cell frequencies were significantly higher, particularly during phases with elevated viral loads and liver enzyme levels. Liver plasma cells exhibited signs of active proliferation, especially in the immune active phase. Our findings suggest a potential role for plasma cells, alongside potential implications and consequences of local proliferation, within the livers of CHB patients. While the significance of AtM B cells remains uncertain, further investigation is warranted to determine their responsiveness to interferons and their role in CHB.
RESUMO
Prior to nuclear export, the hepatitis B virus (HBV) pregenomic RNA may be spliced by the host cell spliceosome to form shorter RNA sequences known as splice variants. Due to deletions in the open reading frames, splice variants may encode novel fusion proteins. Although not essential for HBV replication, the role of splice variants and their novel fusion proteins largely remains unknown. Some splice variants and their encoded novel fusion proteins have been shown to impair or promote wild-type HBV replication in vitro, and although splice variants Sp3 and Sp9 are two of the most common splice variants identified to date, their in vitro replication phenotype and their impact on wild-type HBV replication are unclear. Here, we utilize greater than genome-length Sp3 and Sp9 constructs to investigate their replication phenotype in vitro, and their impact on wild-type HBV replication. We show that Sp3 and Sp9 were incapable of autonomous replication, which was rescued by providing the polymerase and core proteins in trans. Furthermore, we showed that Sp3 had no impact on wild-type HBV replication, whereas Sp9 strongly reduced wild-type HBV replication in co-transfection experiments. Knocking out Sp9 novel precore-surface and core-surface fusion protein partially restored replication, suggesting that these proteins contributed to suppression of wild-type HBV replication, providing further insights into factors regulating HBV replication in vitro. IMPORTANCE: The role of hepatitis B virus (HBV) splice variants in HBV replication and pathogenesis currently remains largely unknown. However, HBV splice variants have been associated with the development of hepatocellular carcinoma, suggesting a role in HBV pathogenesis. Several in vitro co-transfection studies have shown that different splice variants have varying impacts on wild-type HBV replication, perhaps contributing to viral persistence. Furthermore, all splice variants are predicted to produce novel fusion proteins. Sp1 hepatitis B splice protein contributes to liver disease progression and apoptosis; however, the function of other HBV splice variant novel fusion proteins remains largely unknown. We show that Sp9 markedly impairs HBV replication in a cell culture co-transfection model, mediated by expression of Sp9 novel fusion proteins. In contrast, Sp3 had no effect on wild-type HBV replication. Together, these studies provide further insights into viral factors contributing to regulation of HBV replication.
Assuntos
Hepatite B , Neoplasias Hepáticas , Isoformas de Proteínas , Proteínas Virais , Replicação Viral , Humanos , DNA Viral/genética , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Fenótipo , Isoformas de Proteínas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Carcinoma Hepatocelular/virologiaRESUMO
The antiviral role of the tripartite motif-containing (TRIM) protein family , a member of the E3-ubiquitin ligase family, has recently been actively studied. Hepatitis B virus (HBV) infection is a major contributor to liver diseases; however, the host factors regulated by cytokine-inducible TRIM21 to suppress HBV remain unclear. In this study, we showed the antiviral efficacy of TRIM21 against HBV in hepatoma cell lines, primary human hepatocytes isolated from patient liver tissues, and mouse model. Using TRIM21 knock-out cells, we confirmed that the antiviral effects of interferon-gamma, which suppress HBV replication, are diminished when TRIM21 is deficient. Northern blot analysis confirmed a reduction of HBV RNA levels by TRIM21. Using Luciferase reporter assay, we also discovered that TRIM21 decreases the activity of HBV enhancers, which play a crucial role in covalently closed circular DNA transcription. The participation of the RING domain and PRY-SPRY domain in the anti-HBV effect of TRIM21 was demonstrated through experiments using deletion mutants. We identified a novel interaction between TRIM21 and hepatocyte nuclear factor 4α (HNF4α) through co-immunoprecipitation assay. More specifically, ubiquitination assay revealed that TRIM21 promotes ubiquitin-mediated proteasomal degradation of HNF4α. HNF1α transcription is down-regulated as a result of the degradation of HNF4α, an activator for the HNF1α promoter. Therefore, the reduction of key HBV enhancer activators, HNF4α and HNF1α, by TRIM21 resulted in a decline in HBV transcription, ultimately leading to the inhibition of HBV replication.IMPORTANCEDespite extensive research efforts, a definitive cure for chronic hepatitis B remains elusive, emphasizing the persistent importance of this viral infection as a substantial public health concern. Although the risks associated with hepatitis B virus (HBV) infection are well known, host factors capable of suppressing HBV are largely uncharacterized. This study elucidates that tripartite motif-containing protein 21 (TRIM21) suppresses HBV transcription and consequently inhibits HBV replication by downregulating the hepatocyte nuclear factors, which are host factors associated with the HBV enhancers. Our findings demonstrate a novel anti-HBV mechanism of TRIM21 in interferon-gamma-induced anti-HBV activity. These findings may contribute to new strategies to block HBV.
Assuntos
Vírus da Hepatite B , Fator 4 Nuclear de Hepatócito , Hepatócitos , Interferon gama , Ribonucleoproteínas , Replicação Viral , Humanos , Vírus da Hepatite B/fisiologia , Animais , Camundongos , Interferon gama/farmacologia , Interferon gama/metabolismo , Hepatócitos/virologia , Hepatócitos/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hepatite B/virologia , Hepatite B/metabolismo , Células Hep G2 , Linhagem Celular TumoralRESUMO
Chronic hepatitis B virus (HBV) infections are strongly associated with liver cirrhosis, inflammation, and hepatocellular carcinoma. In this context, the viral HBx protein is considered as a major factor influencing HBV-associated pathogenesis through deregulation of multiple cellular signaling pathways and is therefore a potential target for prognostic and therapeutic applications. However, HBV-associated pathogenesis differs significantly between genotypes, with the relevant factors and in particular the contribution of the genetic diversity of HBx being largely unknown. To address this question, we studied the specific genotype-dependent impact of HBx on cellular signaling pathways, focusing in particular on morphological and functional parameters of mitochondria. To exclusively investigate the impact of HBx of different genotypes on integrity and function of mitochondria in the absence of additional viral factors, we overexpressed HBx in Huh7 or HepG2 cells. Key signaling pathways were profiled by kinome analysis and correlated with expression levels of mitochondrial and pathogenic markers. Conclusively, HBx of genotypes A and G caused strong disruption of mitochondrial morphology alongside an induction of PTEN-induced putative kinase 1/Parkin-mediated mitophagy. These effects were only moderately dysregulated by genotypes B and E, whereas genotypes C and D exhibit an intermediate effect in this regard. Accordingly, changes in mitochondrial membrane potential and elevated reactive oxygen species production were associated with the HBx-mediated dysfunction among different genotypes. Also, genotype-related differences in mitophagy induction were identified and indicated that HBx-mediated changes in the mitochondria morphology and function strongly depend on the genotype. This indicates a relevant role of HBx in the process of genotype-dependent liver pathogenesis of HBV infections and reveals underlying mechanisms.IMPORTANCEThe hepatitis B virus is the main cause of chronic liver disease worldwide and differs in terms of pathogenesis and clinical outcome among the different genotypes. Furthermore, the viral HBx protein is a known factor in the progression of liver injury by inducing aberrant mitochondrial structures and functions. Consequently, the selective removal of dysfunctional mitochondria is essential to maintain overall cellular homeostasis and cell survival. Consistent with the intergenotypic difference of HBV, our data reveal significant differences regarding the impact of HBx of different genotypes on mitochondrial dynamic and function and thereby on radical oxygen stress levels within the cell. We subsequently observed that the induction of mitophagy differs significantly across the heterogenetic HBx proteins. Therefore, this study provides evidence that HBx-mediated changes in the mitochondria dynamics and functionality strongly depend on the genotype of HBx. This highlights an important contribution of HBx in the process of genotype-dependent liver pathogenesis.
Assuntos
Vírus da Hepatite B , Dinâmica Mitocondrial , Transdução de Sinais , Transativadores , Proteínas Virais Reguladoras e Acessórias , Humanos , Carcinoma Hepatocelular/virologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Genótipo , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Hepatite B Crônica/metabolismo , Hepatite B Crônica/patologia , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Mitofagia , Espécies Reativas de Oxigênio/metabolismo , Transativadores/metabolismo , Transativadores/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.
Assuntos
Antivirais , Apoptose , Regulação Viral da Expressão Gênica , Antígenos do Núcleo do Vírus da Hepatite B , Vírus da Hepatite B , Hepatócitos , Biossíntese de Proteínas , Animais , Camundongos , Antivirais/farmacologia , Antivirais/uso terapêutico , Apoptose/efeitos dos fármacos , Capsídeo/química , Capsídeo/classificação , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Hepatite B/tratamento farmacológico , Hepatite B/imunologia , Hepatite B/metabolismo , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Camundongos Endogâmicos C57BL , Camundongos SCID , Replicação Viral , Linhagem Celular , Linfócitos T CD8-Positivos/imunologia , Apresentação de AntígenoRESUMO
Liver-specific ten-eleven translocation (Tet) methylcytosine dioxygenases 2 and 3 (Tet2 plus Tet3)-deficient hepatitis B virus (HBV) transgenic mice fail to support viral biosynthesis. The levels of viral transcription and replication intermediates are dramatically reduced. Hepatitis B core antigen is only observed in a very limited number of pericentral hepatocytes in a pattern that is similar to glutamate-ammonia ligase (Glul), a ß-catenin target gene. HBV transcript abundance in adult Tet-deficient mice resembles that observed in wild-type neonatal mice. Furthermore, the RNA levels of several ß-catenin target genes including Glul, Lhpp, Notun, Oat, Slc1a2, and Tbx3 in Tet-deficient mice were also similar to that observed in wild-type neonatal mice. As HBV transcription is regulated by ß-catenin, these findings support the suggestion that neonatal Tet deficiency might limit ß-catenin target gene expression, limiting viral biosynthesis. Additionally, HBV transgene DNA displays increased 5-methylcytosine (5mC) frequency at CpG sequences consistent with neonatal Tet deficiency being responsible for decreased developmental viral DNA demethylation mediated by 5mC oxidation to 5-hydroxymethylcytosine, a process that might be responsible for the reduction in cellular ß-catenin target gene expression and viral transcription and replication.IMPORTANCEChronic hepatitis B virus (HBV) infection causes significant worldwide morbidity and mortality. There are no curative therapies available to resolve chronic HBV infections, and the small viral genome limits molecular targets for drug development. An alternative approach to drug development is to target cellular genes essential for HBV biosynthesis. In the liver, ten-eleven translocation (Tet) genes encode cellular enzymes that are not essential for postnatal mouse development but represent essential activities for viral DNA demethylation and transcription. Consequently, Tet inhibitors may potentially be developed into therapeutic agents capable of inducing and/or maintaining HBV covalently closed circular DNA methylation, resulting in transcriptional silencing and the resolution of chronic viral infection.
Assuntos
Proteínas de Ligação a DNA , Dioxigenases , Vírus da Hepatite B , Animais , Camundongos , beta Catenina/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Desmetilação do DNA , Metilação de DNA , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Vírus da Hepatite B/metabolismo , Camundongos TransgênicosRESUMO
Liver cancer is the third leading cause of cancer-related death worldwide, and hepatocellular carcinoma (HCC) accounts for a relatively large proportion of all primary liver malignancies. Among the several known risk factors, hepatitis B virus (HBV) infection is one of the important causes of HCC. In this study, we demonstrated that the HBV-infected HCC patients could be robustly classified into three clinically relevant subgroups, i.e. Cluster1, Cluster2 and Cluster3, based on consistent differentially expressed mRNAs and proteins, which showed better generalization. The proposed three subgroups showed different molecular characteristics, immune microenvironment and prognostic survival characteristics. The Cluster1 subgroup had near-normal levels of metabolism-related proteins, low proliferation activity and good immune infiltration, which were associated with its good liver function, smaller tumor size, good prognosis, low alpha-fetoprotein (AFP) levels and lower clinical stage. In contrast, the Cluster3 subgroup had the lowest levels of metabolism-related proteins, which corresponded with its severe liver dysfunction. Also, high proliferation activity and poor immune microenvironment in Cluster3 subgroup were associated with its poor prognosis, larger tumor size, high AFP levels, high incidence of tumor thrombus and higher clinical stage. The characteristics of the Cluster2 subgroup were between the Cluster1 and Cluster3 groups. In addition, MCM2-7, RFC2-5, MSH2, MSH6, SMC2, SMC4, NCPAG and TOP2A proteins were significantly upregulated in the Cluster3 subgroup. Meanwhile, abnormally high phosphorylation levels of these proteins were associated with high levels of DNA repair, telomere maintenance and proliferative features. Therefore, these proteins could be identified as potential diagnostic and prognostic markers. In general, our research provided a novel analytical protocol and insights for the robust classification, treatment and prevention of HBV-infected HCC.
Assuntos
Carcinoma Hepatocelular , Hepatite B , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Vírus da Hepatite B/metabolismo , Neoplasias Hepáticas/patologia , alfa-Fetoproteínas/metabolismo , Hepatite B/complicações , Microambiente TumoralRESUMO
Metabolic reprogramming is a hallmark of cancer. The nicotinamide phosphoribosyltransferase (NAMPT)-mediated salvage pathway maintains sufficient cellular NAD levels and is required for tumorigenesis and development. However, the molecular mechanism by which NAMPT contributes to HBV-associated hepatocellular carcinoma (HCC) remains not fully understood. In the present study, our results showed that NAMPT protein was obviously upregulated in HBV-positive HCC tissues compared with HBV-negative HCC tissues. NAMPT was positively associated with aggressive HCC phenotypes and poor prognosis in HBV-positive HCC patients. NAMPT overexpression strengthened the proliferative, migratory, and invasive capacities of HBV-associated HCC cells, while NAMPT-insufficient HCC cells exhibited decreased growth and mobility. Mechanistically, we demonstrated that NAMPT activated SREBP1 (sterol regulatory element-binding protein 1) by increasing the expression and nuclear translocation of SREBP1, leading to the transcription of SREBP1 downstream lipogenesis-related genes and the production of intracellular lipids and cholesterol. Altogether, our data uncovered an important molecular mechanism by which NAMPT promoted HBV-induced HCC progression through the activation of SREBP1-triggered lipid metabolism reprogramming and suggested NAMPT as a promising prognostic biomarker and therapeutic target for HBV-associated HCC patients.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Nicotinamida Fosforribosiltransferase , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Vírus da Hepatite B , Lipogênese , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Nicotinamida Fosforribosiltransferase/genéticaRESUMO
In contrast to horizontal transmission of hepatitis B virus (HBV) between adults, which often leads to self-limited acute infection, vertical transmission of HBV from mother to child often leads to chronic infection. However, the mechanisms linking vertical transmission with chronic infection are not known. We developed a mouse model to study the effect of maternal HBV infection on HBV persistence in offspring and found that HBV carried by the mother impaired CD8(+) T cell responses to HBV in her offspring, resulting in HBV persistence. This impairment of CD8(+) T cell responses was mediated by hepatic macrophages, which were predisposed by maternal HBV e antigen (HBeAg) to support HBV persistence by upregulation of inhibitory ligand PD-L1 and altered polarization upon restimulation with HBeAg. Depletion of hepatic macrophages led to CD8(+) T cell activation and HBV clearance in the offspring, raising the possibility of targeting macrophages to treat chronic HBV patients.
Assuntos
Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Vírus da Hepatite B/fisiologia , Hepatite B/imunologia , Transmissão Vertical de Doenças Infecciosas , Macrófagos/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Animais , Animais Geneticamente Modificados , Antígeno B7-H1/genética , Linfócitos T CD8-Positivos/virologia , Feminino , Regulação da Expressão Gênica , Hepatite B/transmissão , Antígenos E da Hepatite B/imunologia , Humanos , Ativação Linfocitária , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Carga ViralRESUMO
The effects of the obliteration of portal venules (OPV) in cirrhotic portal hypertension are poorly understood. To investigate its contribution to portal hypertension in biliary cirrhosis and its underlying mechanism, we evaluated OPV using two-dimensional (2D) histopathology in liver explants from patients with biliary atresia (BA, n = 63), primary biliary cholangitis (PBC, n = 18), and hepatitis B-related cirrhosis (Hep-B-cirrhosis, n = 35). Then, three-dimensional (3D) OPV was measured by X-ray phase-contrast CT in two parallel models in rats following bile duct ligation (BDL) or carbon tetrachloride (CCl4) administration, representing biliary cirrhosis and post-necrotic cirrhosis, respectively. The portal pressure was also measured in the two models. Finally, the effects of proliferative bile ducts on OPV were investigated. We found that OPV was significantly more frequent in patients with biliary cirrhosis, including BA (78.57 ± 16.45%) and PBC (60.00 ± 17.15%), than that in Hep-B-cirrhotic patients (29.43 ± 14.94%, p < 0.001). OPV occurred earlier, evidenced by the paired liver biopsy at a Kasai procedure (KP), and was irreversible even after a successful KP in the patients with BA. OPV was also significantly more frequent in the BDL models than in the CCl4 models, as shown by 2D and 3D quantitative analysis. Portal pressure was significantly higher in the BDL model than that in the CCl4 model. With the proliferation of bile ducts, portal venules were compressed and irreversibly occluded, contributing to the earlier and higher portal pressure in biliary cirrhosis. OPV, as a pre-sinusoidal component, plays a key role in the pathogenesis of portal hypertension in biliary cirrhosis. The proliferated bile ducts and ductules gradually take up the 'territory' originally attributed to portal venules and compress the portal venules, which may lead to OPV in biliary cirrhosis. © 2024 The Pathological Society of Great Britain and Ireland.
Assuntos
Hipertensão Portal , Cirrose Hepática Biliar , Veia Porta , Hipertensão Portal/patologia , Hipertensão Portal/fisiopatologia , Animais , Cirrose Hepática Biliar/patologia , Cirrose Hepática Biliar/complicações , Cirrose Hepática Biliar/fisiopatologia , Masculino , Humanos , Feminino , Veia Porta/patologia , Vênulas/patologia , Ratos , Adulto , Pressão na Veia Porta , Pessoa de Meia-Idade , Modelos Animais de Doenças , Fígado/patologia , Fígado/irrigação sanguínea , Ratos Sprague-Dawley , Ductos Biliares/patologia , Adulto Jovem , AdolescenteRESUMO
The question of whether patients in the immune-tolerant (IT) phase of chronic hepatitis B virus (HBV) infection should undergo antiviral therapy and determine the optimal regimen remains unclear. A comprehensive search of PubMed, Embase, MEDLINE, Cochrane Library, and Wanfang Data from inception to 5 December 2023, was conducted. Studies reporting on key outcomes such as HBV DNA undetectability, HBeAg loss or seroconversion, HBsAg loss or seroconversion, and hepatocellular carcinoma (HCC) incidence in patients in the IT phase of chronic HBV infection were included. In total, 23 studies were incorporated. Approximately 4% of patients in the IT phase achieved spontaneous HBeAg loss over 48 weeks of follow-up. Antiviral therapy demonstrated a favourable impact on HBV DNA negative conversion (Children: risk ratios [RR] = 6.83, 95% CI: 2.90-16.05; Adults: RR = 25.84, 95% CI: 6.47-103.31) and HBsAg loss rates (Children: RR = 9.49, 95% CI: 1.74-51.76; Adults: RR = 7.35, 95% CI: 1.41-38.27) for patients in the IT phase. Subgroup analysis revealed that in adult patients in the IT phase, interferon plus nucleos(t)ide analogues (NA)-treated patients exhibited a higher pooled rate of HBsAg loss or seroconversion than those treated with NA monotherapy (9% vs. 0%). Additionally, the pooled annual HCC incidence for patients in the IT phase was 3.03 cases per 1000 person-years (95% CI: 0.99-5.88). Adult patients in the IT phase had a significantly lower HCC incidence risk than HBeAg-positive indeterminate phase patients (RR = 0.46, 95% CI: 0.32-0.66), with no significant differences observed between IT and immune-active phases. Presently, there is insufficient evidence solely based on reducing the risk of HCC incidence, to recommend treating patients in the IT phase of chronic HBV infection. However, both adult and paediatric patients in the IT phase responded well to antiviral therapy, showing favourable rates of HBsAg loss or seroconversion.
Assuntos
Antivirais , Carcinoma Hepatocelular , Antígenos E da Hepatite B , Hepatite B Crônica , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/virologia , Carcinoma Hepatocelular/imunologia , Hepatite B Crônica/complicações , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/imunologia , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/virologia , Neoplasias Hepáticas/imunologia , Antivirais/uso terapêutico , Antígenos E da Hepatite B/sangue , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Incidência , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/imunologia , DNA Viral/sangue , Tolerância Imunológica , Resultado do Tratamento , SoroconversãoRESUMO
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.