RESUMO
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
Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.
Assuntos
Ácidos e Sais Biliares , Microscopia Crioeletrônica , Vírus da Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Receptores Virais , Simportadores , Anticorpos , Ácidos e Sais Biliares/metabolismo , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Receptores Virais/química , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestruturaRESUMO
The liver takes up bile salts from blood to generate bile, enabling absorption of lipophilic nutrients and excretion of metabolites and drugs1. Human Na+-taurocholate co-transporting polypeptide (NTCP) is the main bile salt uptake system in liver. NTCP is also the cellular entry receptor of human hepatitis B and D viruses2,3 (HBV/HDV), and has emerged as an important target for antiviral drugs4. However, the molecular mechanisms underlying NTCP transport and viral receptor functions remain incompletely understood. Here we present cryo-electron microscopy structures of human NTCP in complexes with nanobodies, revealing key conformations of its transport cycle. NTCP undergoes a conformational transition opening a wide transmembrane pore that serves as the transport pathway for bile salts, and exposes key determinant residues for HBV/HDV binding to the outside of the cell. A nanobody that stabilizes pore closure and inward-facing states impairs recognition of the HBV/HDV receptor-binding domain preS1, demonstrating binding selectivity of the viruses for open-to-outside over inward-facing conformations of the NTCP transport cycle. These results provide molecular insights into NTCP 'gated-pore' transport and HBV/HDV receptor recognition mechanisms, and are expected to help with development of liver disease therapies targeting NTCP.
Assuntos
Ácidos e Sais Biliares , Microscopia Crioeletrônica , Fígado , Transportadores de Ânions Orgânicos Dependentes de Sódio , Sódio , Simportadores , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Vírus da Hepatite B/metabolismo , Vírus Delta da Hepatite/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Conformação Proteica , Receptores Virais/metabolismo , Anticorpos de Domínio Único , Sódio/metabolismo , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestrutura , Internalização do VírusRESUMO
Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus-transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.
Assuntos
Microscopia Crioeletrônica , Vírus da Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Receptores Virais , Simportadores , Animais , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Bovinos , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Humanos , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Ratos , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Sódio/metabolismo , Simportadores/química , Simportadores/genética , Simportadores/metabolismo , Simportadores/ultraestruturaRESUMO
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
Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter (BCP). Here we show that the hypoxic-dependent increase in BCP-derived transcripts is dependent on N6-methyladenosine (m6A) modifications in the 5' stem loop that regulate RNA half-life. Application of a probe-enriched long-read sequencing method to accurately map the HBV transcriptome showed an increased abundance of pre-genomic RNA under hypoxic conditions. Mapping the transcription start sites of BCP-RNAs identified a role for hypoxia to regulate pre-genomic RNA splicing that is dependent on m6A modification. Bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2-NTCP cell line showed increased ALKBH5 gene expression under hypoxic conditions and a concomitant reduction in m6A-modified HBV BCP-RNA and host RNAs. Silencing the demethylase reduced the level of BCP-RNAs and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.
Assuntos
Vírus da Hepatite B , Hepatite B , Animais , Humanos , Camundongos , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Fucosiltransferases/genética , Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hipóxia , Oxigênio , RNA , TranscriptomaRESUMO
Members of the serine-arginine protein kinase (SRPK) family, SRPK1 and SRPK2, phosphorylate the hepatitis B core protein (Cp) and are crucial for pregenomic RNA encapsidation during viral nucleocapsid assembly. Among them, SRPK2 exhibits higher kinase activity toward Cp. In this study, we identified Cp sites that are phosphorylated by SRPK2 and demonstrated that the kinase utilizes an SRPK-specific docking groove to interact with and regulate the phosphorylation of the C-terminal arginine rich domain of Cp. We determined that direct interaction between the docking groove of SRPK2 and unphosphorylated Cp inhibited premature viral capsid assembly in vitro, whereas the phosphorylation of the viral protein reactivated the process. Pull-down assays together with the new cryo-electron microscopy structure of the HBV capsid in complex with SRPK2 revealed that the kinases decorate the surface of the viral capsid by interacting with the C-terminal domain of Cp, underscoring the importance of the docking interaction in regulating capsid assembly and pregenome packaging. Moreover, SRPK2-knockout in HepG2 cells suppressed Cp phosphorylation, indicating that SRPK2 is an important cellular kinase for HBV life cycle.
Assuntos
Capsídeo , Vírus da Hepatite B , Fosforilação , Capsídeo/metabolismo , Vírus da Hepatite B/metabolismo , Microscopia Crioeletrônica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas do Capsídeo/metabolismo , Montagem de Vírus/fisiologia , Arginina/metabolismoRESUMO
Hepatitis B virus (HBV) chronically infects 296 million people worldwide, posing a major global health threat. Export of HBV RNAs from the nucleus to the cytoplasm is indispensable for viral protein translation and genome replication, however the mechanisms regulating this critical process remain largely elusive. Here, we identify a key host factor embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) that binds HBV RNAs and controls their nuclear export. Using an unbiased quantitative proteomics screen, we demonstrate direct binding of ELAVL1 to the HBV pregenomic RNA (pgRNA). ELAVL1 knockdown inhibits HBV RNAs posttranscriptional regulation and suppresses viral replication. Further mechanistic studies reveal ELAVL1 recruits the nuclear export receptor CRM1 through ANP32A and ANP32B to transport HBV RNAs to the cytoplasm via specific AU-rich elements, which can be targeted by a compound CMLD-2. Moreover, ELAVL1 protects HBV RNAs from DIS3+RRP6+ RNA exosome mediated nuclear RNA degradation. Notably, we find HBV core protein is dispensable for HBV RNA-CRM1 interaction and nuclear export. Our results unveil ELAVL1 as a crucial host factor that regulates HBV RNAs stability and trafficking. By orchestrating viral RNA nuclear export, ELAVL1 is indispensable for the HBV life cycle. Our study highlights a virus-host interaction that may be exploited as a new therapeutic target against chronic hepatitis B.
Assuntos
Vírus da Hepatite B , RNA Viral , Animais , Humanos , Vírus da Hepatite B/metabolismo , Transporte Ativo do Núcleo Celular , RNA Viral/genética , RNA Viral/metabolismo , Drosophila/genética , Replicação Viral/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismoRESUMO
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
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
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 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
Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.
Assuntos
Carcinoma Hepatocelular , Hepatite B , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Transativadores/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteína bcl-X/química , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatite B/complicações , Hepatite B/patologiaRESUMO
As an important protein encoded by hepatitis B virus (HBV), HBV X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). It has been shown that seven in absentia homologue 1 (SIAH1) could regulates the degradation of HBx through the ubiquitin-proteasome pathway. However, as a member of SIAH family, the regulatory effects of SIAH2 on HBx remain unclear. In this study, we first confirmed that SIAH2 could reduce the protein levels of HBx depending on its E3 ligase activity. Moreover, SIAH2 interacted with HBx and induced its K48-linked polyubiquitination and proteasomal degradation. Furthermore, we provided evidence that SIAH2 inhibits HBx-associated HCC cells proliferation by regulating HBx. In conclusion, our study identified a novel role for SIAH2 in promoting HBx degradation and SIAH2 exerts an inhibitory effect in the proliferation of HBx-associated HCC through inducing the degradation of HBx. Our study provides a new idea for the targeted degradation of HBx and may have great huge significance into providing novel evidence for the targeted therapy of HBV-infected HCC.
Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Vírus da Hepatite B , Neoplasias Hepáticas , Proteínas Nucleares , Proteólise , Transativadores , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteínas Virais Reguladoras e Acessórias , Humanos , Proteínas Virais Reguladoras e Acessórias/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Transativadores/metabolismo , Transativadores/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Neoplasias Hepáticas/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Linhagem Celular Tumoral , Transdução de Sinais , Células Hep G2RESUMO
Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/ß complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impß as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T = 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0 Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impß-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.
Assuntos
Capsídeo , Vírus da Hepatite B , Humanos , beta Carioferinas , Capsídeo/química , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , Hepatite B/virologia , Vírus da Hepatite B/metabolismo , Montagem de VírusRESUMO
Hepatitis B virus (HBV) is a hepatotropic DNA virus that has a very compact genome. Due to this genomic density, several distinct mechanisms are used to facilitate the viral life cycle. Recently, accumulating evidence show that G-quadruplex (G4) in different viruses play essential regulatory roles in key steps of the viral life cycle. Although G4 structures in the HBV genome have been reported, their function in HBV replication remains elusive. In this study, we treated an HBV replication-competent cell line and HBV-infected cells with the G4 structure stabilizer pyridostatin (PDS) and evaluated different HBV replication markers to better understand the role played by the G4. In both models, we found PDS had no effect on viral precore RNA (pcRNA) or pre-genomic RNA (pgRNA), but treatment did increase HBeAg/HBc ELISA reads and intracellular levels of viral core/capsid protein (HBc) in a dose-dependent manner, suggesting post-transcriptional regulation. To further dissect the mechanism of G4 involvement, we used in vitro-synthesized HBV pcRNA and pgRNA. Interestingly, we found PDS treatment only enhanced HBc expression from pgRNA but not HBeAg expression from pcRNA. Our bioinformatic analysis and CD spectroscopy revealed that pgRNA harbors a conserved G4 structure. Finally, we introduced point mutations in pgRNA to disrupt its G4 structure and observed the resulting mutant failed to respond to PDS treatment and decreased HBc level in in vitro translation assay. Taken together, our data demonstrate that HBV pgRNA contains a G4 structure that plays a vital role in the regulation of viral mRNA translation.
Assuntos
Quadruplex G , Vírus da Hepatite B , Hepatite B , Humanos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Hepatite B/virologia , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo , Replicação Viral/genética , Linhagem Celular , Quadruplex G/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Mutação , Aminoquinolinas/farmacologiaRESUMO
There are approximately 250 million people chronically infected with hepatitis B virus (HBV) worldwide. Although HBV is often integrated into the host genome and promotes hepatocarcinogenesis, vulnerability of HBV integration in liver cancer cells has not been clarified. The aim of our study is to identify vulnerability factors for HBV-associated hepatocarcinoma. Loss-of-function screening was undertaken in HepG2 and HBV-integrated HepG2.2.15 cells expressing SpCas9 using a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library. Genes whose guide RNA (gRNA) abundance significantly decreased in HepG2.2.15 cells but not in HepG2 cells were extracted using the MAGeCK algorithm. We identified four genes (BCL2L1, VPS37A, INSIG2, and CFLAR) that showed significant reductions of gRNA abundance and thus potentially involved in the vulnerability of HBV-integrated cancer cells. Among them, siRNA-mediated mRNA inhibition or CRISPR-mediated genetic deletion of INSIG2 significantly impaired cell proliferation in HepG2.2.15 cells but not in HepG2 cells. Its inhibitory effect was alleviated by cotransfection of siRNAs targeting HBV. INSIG2 inhibition suppressed the pathways related to cell cycle and DNA replication, downregulated cyclin-dependent kinase 2 (CDK2) levels, and delayed the G1 -to-S transition in HepG2.2.15 cells. CDK2 inhibitor suppressed cell cycle progression in HepG2.2.15 cells and INSIG2 inhibition did not suppress cell proliferation in the presence of CDK2 inhibitor. In conclusion, INSIG2 inhibition induced cell cycle arrest in HBV-integrated hepatoma cells in a CDK2-dependent manner, and thus INSIG2 might be a vulnerability factor for HBV-associated liver cancer.
Assuntos
Carcinoma Hepatocelular , Hepatite B , Neoplasias Hepáticas , Humanos , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Carcinoma Hepatocelular/genética , RNA Guia de Sistemas CRISPR-Cas , Neoplasias Hepáticas/genética , Linhagem Celular , Células Hep G2 , RNA Interferente Pequeno/metabolismo , Replicação Viral/genética , Hepatite B/genética , DNA Viral/genética , Proteínas de Membrana/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
IMPORTANCE: The biogenesis and clinical application of serum HBV pgRNA have been a research hotspot in recent years. This study further characterized the heterogeneity of the 3' terminus of capsid RNA by utilizing a variety of experimental systems conditionally supporting HBV genome replication and secretion, and reveal that the 3' truncation of capsid pgRNA is catalyzed by cellular ribonuclease(s) and viral RNaseH at positions after and before 3' DR1, respectively, indicating the 3' DR1 as a boundary between the encapsidated portion of pgRNA for reverse transcription and the 3' unprotected terminus, which is independent of pgRNA length and the 3' terminal sequence. Thus, our study provides new insights into the mechanism of pgRNA encapsidation and reverse transcription, as well as the optimization of serum HBV RNA diagnostics.
Assuntos
Capsídeo , Genoma Viral , Vírus da Hepatite B , RNA Viral , Replicação Viral , Capsídeo/metabolismo , Genoma Viral/genética , Hepatite B/diagnóstico , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/metabolismo , Transcrição Reversa , Ribonuclease H/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral/genéticaRESUMO
Hepatitis B virus (HBV) chronically infects approximately 300 million people worldwide, and permanently repressing transcription of covalently closed circular DNA (cccDNA), the episomal viral DNA reservoir, is an attractive approach toward curing HBV. However, the mechanism underlying cccDNA transcription is only partially understood. In this study, by illuminating cccDNA of wild-type HBV (HBV-WT) and transcriptionally inactive HBV that bears a deficient HBV X gene (HBV-ΔX), we found that the HBV-ΔX cccDNA more frequently colocalizes with promyelocytic leukemia (PML) bodies than that of HBV-WT cccDNA. A small interfering RNA (siRNA) screen targeting 91 PML body-related proteins identified SMC5-SMC6 localization factor 2 (SLF2) as a host restriction factor of cccDNA transcription, and subsequent studies showed that SLF2 mediates HBV cccDNA entrapment in PML bodies by interacting with the SMC5/6 complex. We further showed that the region of SLF2 comprising residues 590 to 710 interacts with and recruits the SMC5/6 complex to PML bodies, and the C-terminal domain of SLF2 containing this region is necessary for repression of cccDNA transcription. Our findings shed new light on cellular mechanisms that inhibit HBV infection and lend further support for targeting the HBx pathway to repress HBV activity. IMPORTANCE Chronic HBV infection remains a major public health problem worldwide. Current antiviral treatments rarely cure the infection, as they cannot clear the viral reservoir, cccDNA, in the nucleus. Therefore, permanently silencing HBV cccDNA transcription represents a promising approach for a cure of HBV infection. Our study provides new insights into the cellular mechanisms that restrict HBV infection, revealing the role of SLF2 in directing HBV cccDNA to PML bodies for transcriptional repression. These findings have important implications for the development of antiviral therapies against HBV.