RESUMO
Hepatitis B virus (HBV) infection is a serious global health problem. After the viruses infect the human body, the host can respond to the virus infection by coordinating various cellular responses, in which mitochondria play an important role. Evidence has shown that mitochondrial proteins are involved in host antiviral responses. In this study, we found that the overexpression of TIM22 and TIM29, the members of the inner membrane translocase TIM22 complex, significantly reduced the level of intracellular HBV DNA and RNA and secreted HBV surface antigens and E antigen. The effects of TIM22 and TIM29 on HBV replication and transcription is attributed to the reduction of core promoter activity mediated by the increased expression of SRSF1 which acts as a suppressor of HBV replication. This study provides new evidence for the critical role of mitochondria in the resistance of HBV infection and new targets for the development of treatment against HBV infection.
Assuntos
Vírus da Hepatite B , Hepatite B , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Fatores de Processamento de Serina-Arginina , Humanos , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Fatores de Processamento de Serina-Arginina/metabolismo , Replicação Viral , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismoRESUMO
Chronic hepatitis B virus (HBV) infection can lead to fibrosis, liver cirrhosis, and primary hepatocellular carcinoma. Investigating host factors that regulate HBV replication helps to identify antiviral targets. In the current study, we identified Nicotinamide N-Methyltransferase gene (NNMT) as a novel factor that regulates HBV transcription. NNMT is up-regulated at both the mRNA and protein levels in HepG2.2.15 cells compared to HepG2 cells. Overexpression of NNMT reduces HBV replication in several cell models, while knockdown of NNMT enhances HBV DNA levels. Mechanistically, NNMT suppresses HBV DNA replication by inhibiting HBV RNA transcription. The region required for the inhibitory effect of NNMT was narrowed to nt 1672-1708 in enhancer II by luciferase assays. On the other hand, ChIP assays and EMSA results showed that NNMT does not bind to this region substantially, either directly or indirectly. Next, a collection of hepatic nuclear receptor transcription factors was screened to determine whether they were affected by NNMT overexpression. NR5A1, a positive regulator of HBV replication, decreased significantly after NNMT overexpression. Collectively, the findings of this study shed light on the regulation of HBV transcription.
Assuntos
Carcinoma Hepatocelular , Hepatite B Crônica , Neoplasias Hepáticas , Vírus da Hepatite B/fisiologia , Humanos , Neoplasias Hepáticas/genética , Nicotinamida N-Metiltransferase/metabolismo , Fator Esteroidogênico 1 , Replicação ViralRESUMO
BACKGROUND: Coronavirus disease 2019 (COVID-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. Little is known about the longitudinal dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) in patients with COVID-19. METHODS: Blood samples (n = 173) were collected from 30 patients with COVID-19 over a 3-month period after symptom onset and analyzed for SARS-CoV-2-specific NAbs using the lentiviral pseudotype assay, coincident with the levels of IgG and proinflammatory cytokines. RESULTS: SARS-CoV-2-specific NAb titers were low for the first 7-10 days after symptom onset and increased after 2-3 weeks. The median peak time for NAbs was 33 days (interquartile range [IQR], 24-59 days) after symptom onset. NAb titers in 93.3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34.8% (IQR, 19.6-42.4%). NAb titers increased over time in parallel with the rise in immunoglobulin G (IgG) antibody levels, correlating well at week 3 (r = 0.41, P < .05). The NAb titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including stem cell factor (SCF), TNF-related apoptosis-inducing ligand (TRAIL), and macrophage colony-stimulating factor (M-CSF). CONCLUSIONS: These data provide useful information regarding dynamic changes in NAbs in patients with COVID-19 during the acute and convalescent phases.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , PandemiasRESUMO
BACKGROUND & AIMS: Current antiviral therapies help keep HBV under control, but they are not curative, as they are unable to eliminate the intracellular viral replication intermediate termed covalently closed circular DNA (cccDNA). Therefore, there remains an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx. METHODS: We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model. RESULTS: Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells. CONCLUSIONS: Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability. LAY SUMMARY: Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.
Assuntos
Antivirais/administração & dosagem , DNA Circular/metabolismo , Dicumarol/administração & dosagem , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/metabolismo , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/metabolismo , Proteólise/efeitos dos fármacos , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais Reguladoras e Acessórias/metabolismo , Animais , DNA Circular/isolamento & purificação , Modelos Animais de Doenças , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NAD(P)H Desidrogenase (Quinona)/genética , Transfecção , Resultado do Tratamento , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Chronic hepatitis B virus (HBV) infection is a significant public health burden worldwide. HBV covalently closed circular DNA (cccDNA) organized as a minichromosome in nucleus is responsible for viral persistence and is the key obstacle for a cure of chronic hepatitis B (CHB). Recent studies suggest cccDNA transcription is epigenetically regulated by histone modifications, especially histone acetylation and methylation. In the present study, we identified transcriptionally active histone succinylation (H3K122succ) as a new histone modification on cccDNA minichromosome by using cccDNA ChIP-Seq approach. Silent mating type information regulation 2 homolog 7 (SIRT7), as an NAD+-dependent histone desuccinylase, could bind to cccDNA through interaction with HBV core protein where it catalyzed histone 3 lysine 122 (H3K122) desuccinylation. Moreover, SIRT7 acts cooperatively with histone methyltransferase, suppressor of variegation 3-9 homolog 1 (SUV39H1) and SET domain containing 2 (SETD2) to induce silencing of HBV transcription through modulation of chromatin structure. Our data improved the understanding of histone modifications of the cccDNA minichromosome, thus transcriptional silencing of cccDNA may represent a novel antiviral strategy for the prevention or treatment of HBV infection.
Assuntos
Catálise , DNA Circular/metabolismo , Histona Metiltransferases/genética , Histonas/metabolismo , Sirtuínas/metabolismo , DNA Viral/genética , Hepatite B/prevenção & controle , Hepatite B/terapia , Hepatite B/virologia , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/prevenção & controle , Humanos , Sirtuínas/genética , Transcrição Gênica/genética , Replicação Viral/genéticaRESUMO
Hepatitis B virus (HBV) infection is a common infectious disease, in which nuclear covalently closed circular DNA (cccDNA) plays a key role in viral persistence, viral reactivation after treatment withdrawal, and drug resistance. A recent genome-wide association study has identified that the ubiquitin conjugating enzyme E2 L3 (UBE2L3) gene is associated with increased susceptibility to chronic HBV (CHB) infection in adults. However, the association between UBE2L3 and children with CHB and the underlying mechanism remain unclear. In this study, we performed two-stage case-control studies including adults and independent children in the Chinese Han population. The rs59391722 allele in the promoter of the UBE2L3 gene was significantly associated with HBV infection in both adults and children, and it increased the promoter activity of UBE2L3. Serum UBE2L3 protein levels were positively correlated with HBV viral load and hepatitis B e antigen (HBeAg) levels in children with CHB. In an HBV infection cell model, UBE2L3 knockdown significantly reduced total HBV RNAs, 3.5-kb RNA, as well as cccDNA in HBV-infected HepG2-Na+ /taurocholate cotransporting polypeptide cells and human primary hepatocytes. A mechanistic study found that UBE2L3 maintained cccDNA stability by inducing proteasome-dependent degradation of apolipoprotein B mRNA editing enzyme catalytic subunit 3A, which is responsible for the degradation of HBV cccDNA. Moreover, interferon-α (IFN-α) treatment markedly decreased UBE2L3 expression, while UBE2L3 silencing reinforced the antiviral activity of IFN-α on HBV RNAs, cccDNA, and DNA. rs59391722 in UBE2L3 was correlated with HBV DNA suppression and HBeAg loss in response to IFN-α treatment of children with CHB. Conclusion: These findings highlight a host gene, UBE2L3, contributing to the susceptibility to persistent HBV infection; UBE2L3 may be involved in IFN-mediated viral suppression and serve as a potential target in the prevention and treatment of HBV infection.
Assuntos
Citidina Desaminase/metabolismo , Hepatite B Crônica/genética , Enzimas de Conjugação de Ubiquitina/genética , Desaminases APOBEC , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , DNA Circular , Predisposição Genética para Doença , Células Hep G2 , Hepatite B Crônica/tratamento farmacológico , Humanos , Lactente , Interferon-alfa/uso terapêutico , Polimorfismo de Nucleotídeo Único , Enzimas de Conjugação de Ubiquitina/metabolismo , Replicação ViralRESUMO
BACKGROUND: Several studies have focused on the association between KIF1B rs17401966 polymorphism and susceptibility to hepatitis B virus-related (HBV-related) hepatocellular carcinoma (HCC), but the conclusions have been inconsistent. We have conducted this updated meta-analysis to explore the association between KIF1B rs17401966 polymorphism and HCC susceptibility. METHODS: Eligible studies were identified through systematic searches in PubMed, OVID, ISI Web of Science, Chinese National Knowledge Infrastructure, and Wanfang databases. The quality of evidence was systematically assessed by use of the Newcastle-Ottawa Scale for case control studies in meta-analyses. RESULTS: Ten studies containing 18 independent case-control studies were included. The results revealed a significant association between KIF1B rs17401966 polymorphism and susceptibility to HCC under a random-effect allelic model (OR = 0.85, 95% CI 0.76-0.94, P = 0.003); HBV-positive subgroup (OR = 0.82, 95% CI 0.72-0.95, P = 0.007); and Chinese-subgroup (OR = 0.82, 95% CI 0.72-0.93, P = 0.002). CONCLUSIONS: G-allele appears to be a protective allele of KIF1B for HCC, especially in HBV-positive and Chinese populations. More well-designed studies with larger sample size and various ethnic groups and risk factors are needed to establish that KIF1B rs17401966 polymorphism is significantly associated with risk of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Predisposição Genética para Doença , Cinesinas/genética , Neoplasias Hepáticas/genética , Polimorfismo de Nucleotídeo Único , HumanosRESUMO
Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA), which serves as a template for HBV RNA transcription, is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified silent mating type information regulation 2 homolog 3 (SIRT3) as a host factor restricting HBV transcription and replication by screening seven members of the sirtuin family, which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA, as well as replicative intermediate DNA in HBV-infected HepG2-Na+ /taurocholate cotransporting polypeptide cells and primary human hepatocytes. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. A mechanistic study found that nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9. Importantly, occupancy of SIRT3 on cccDNA could increase the recruitment of histone methyltransferase suppressor of variegation 3-9 homolog 1 to cccDNA and decrease recruitment of SET domain containing 1A, leading to a marked increase of trimethyl-histone H3 (Lys9) and a decrease of trimethyl-histone H3 (Lys4) on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor Yin Yang 1 to cccDNA. Finally, hepatitis B viral X protein could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. CONCLUSION: SIRT3 is a host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase; these data provide a rationale for the use of SIRT3 activators in the prevention or treatment of HBV infection. (Hepatology 2018).
Assuntos
DNA Viral/genética , Epigênese Genética/genética , Hepatite B/genética , Domínios PR-SET/genética , Sirtuína 3/genética , Replicação Viral/genética , DNA Complementar/genética , Hepatite B/fisiopatologia , Vírus da Hepatite B/genética , Histona Metiltransferases/metabolismo , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e EspecificidadeRESUMO
The capsid of the hepatitis B virus is an attractive antiviral target for developing therapies against chronic hepatitis B infection. Currently available core protein allosteric modulators (CpAMs) mainly affect one of the two major types of protein-protein interactions involved in the process of capsid assembly, namely, the interaction between the core dimers. Compounds targeting the interaction between two core monomers have not been rigorously screened due to the lack of screening models. We report here a cell-based assay in which the formation of core dimers is indicated by split luciferase complementation (SLC). Making use of this model, 2 compounds, Arbidol (umifenovir) and 20-deoxyingenol, were identified from a library containing 672 compounds as core dimerization regulators. Arbidol and 20-deoxyingenol inhibit the hepatitis B virus (HBV) DNA replication in vitro by decreasing and increasing the formation of core dimer and capsid, respectively. Our results provided a proof of concept for the cell model to be used to screen new agents targeting the step of core dimer and capsid formation.
Assuntos
Antivirais/farmacologia , Diterpenos/farmacologia , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/efeitos dos fármacos , Indóis/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas do Core Viral/antagonistas & inibidores , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , DNA Viral/biossíntese , DNA Viral/genética , Genes Reporter , Células HEK293 , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Ensaios de Triagem em Larga Escala , Humanos , Luciferases/genética , Luciferases/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismoRESUMO
The achievement of a functional cure for chronic hepatitis B (CHB) remains limited to a minority of patients treated with currently approved drugs. The primary objective in developing new anti-HBV drugs is to enhance the functional cure rates for CHB. A critical prerequisite for the functional cure of CHB is a substantial reduction, or even eradication of covalently closed circular DNA (cccDNA). Within this context, the changes in cccDNA levels during treatment become as a pivotal concern. We have previously analyzed the factors influencing cccDNA dynamics and introduced a preliminary classification of hepatitis B treatment strategies based on these dynamics. In this review, we employ a systems thinking perspective to elucidate the fundamental aspects of the HBV replication cycle and to rationalize the classification of treatment strategies according to their impact on the dynamic equilibrium of cccDNA. Building upon this foundation, we categorize current anti-HBV strategies into two distinct groups and advocate for their combined use to significantly reduce cccDNA levels within a well-defined timeframe.
Assuntos
Hepatite B Crônica , Hepatite B , Humanos , Hepatite B Crônica/tratamento farmacológico , Vírus da Hepatite B/genética , DNA Circular/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , DNA Viral/genética , Replicação Viral/genéticaRESUMO
OBJECTIVE: To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate. METHODS: Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis. RESULTS: Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained. CONCLUSIONS: A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.
Assuntos
Linhagem Celular , Replicação do DNA , DNA Viral/biossíntese , Vírus da Hepatite B/genética , Hepatócitos/citologia , Clonagem Molecular , Vetores Genéticos , Células Hep G2 , Hepatócitos/virologia , Humanos , Plasmídeos , DNA Polimerase Dirigida por RNA/genética , Replicação Viral/genéticaRESUMO
OBJECTIVE: To investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system. METHODS: The PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein. RESULTS: The transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein. CONCLUSION: Soluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.
Assuntos
Proteínas Recombinantes/genética , Proteínas do Core Viral/genética , Escherichia coli/metabolismo , Vetores Genéticos , Hepacivirus , Proteínas Recombinantes/metabolismo , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
Eradication of cccDNA is an ideal goal of chronic hepatitis B (CHB) therapy. Understanding the changes in the cccDNA pool during therapy provides a basis for developing CHB treatment strategies. On the other hand, the shift in the balance of the cccDNA pool following therapies allowed researchers to investigate the dynamics of cccDNA. Central to the description of cccDNA dynamics is a parameter called cccDNA half-life. CccDNA half-life is not an intrinsic property of cccDNA molecules, but a description of an observed phenomenon characterized by cccDNA pool decline. Since cccDNA has to be in the nuclei of host cells to function, the half-life of cccDNA is determined by the state and destiny of the host cells. The major factors that drive cccDNA decay include noncytopathic effects and hepatocyte turnover (death and division). In some cases, the determining factor is not the half-life of cccDNA itself, but rather the half-life of the hepatocyte. The main purpose of this review is to analyze the major factors affecting cccDNA half-life and determine the areas requiring further study. In addition, the discrepancy in cccDNA half-life between short-term and long-term nucleot(s)ide analog (NUC) therapy was reported. Hypotheses were proposed to explain the multi-phasic decline of cccDNA during NUC therapy, and a framework based on cccDNA dynamics was suggested for the consideration of various anti-HBV strategies.
RESUMO
Biotin proximity labeling is a technique based on the TurboID enzyme that can be used to capture weak or dynamic interactions that had previously not been used to map proteins interacting with a specific DNA sequence. Here, we present a protocol for identifying specific DNA-sequence-binding proteins. We describe steps for biotin labeling of DNA-binding proteins, protein enrichment and sodium dodecyl sulfate polyacrylamide gel electrophoresis separation, and proteomic analysis. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022).1.
Assuntos
Biotina , Proteínas de Ligação a DNA , Proteômica/métodosRESUMO
Phenotypic assays of hepatitis B virus (HBV) play an important role in research related to the problem of drug resistance that emerges during long-term nucleot(s)ide therapy in patients with chronic hepatitis B. Most of the phenotypic assay systems that are available currently rely on the transfection of recombinant replication-competent HBV DNA into hepatoma cell lines. Cloning clinical HBV isolates using conventional digestion-and-ligation techniques to generate replication-competent recombinants can be very difficult because of the sequence heterogeneity and unique structure of the HBV genome. In this study, a new strategy for constructing an HBV 1.1× recombinant was developed. The core of this strategy is the "fragment substitution reaction" (FSR). FSR allows PCR fragments to be cloned without digestion or ligation, providing a new tool for cloning fragments or genomes amplified from serum HBV DNA, and therefore making the assay of HBV phenotypes more convenient. Using this strategy, a phenotypic assay was performed on an HBV strain carrying an rtS246T variant isolated from a patient with chronic hepatitis B that was only responsive partially to entecavir therapy. The results indicated that this strain is sensitive to entecavir in vitro.
Assuntos
Antivirais/uso terapêutico , Clonagem Molecular/métodos , Farmacorresistência Viral , Vírus da Hepatite B/efeitos dos fármacos , Mutação de Sentido Incorreto , DNA Polimerase Dirigida por RNA/metabolismo , Virologia/métodos , Adulto , Sequência de Bases , DNA Viral/química , DNA Viral/genética , Guanina/análogos & derivados , Guanina/uso terapêutico , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fenótipo , DNA Polimerase Dirigida por RNA/genética , Recombinação Genética , Análise de Sequência de DNARESUMO
Translation elongation factor 1A (eEF1A) is a factor critically involved in the process of protein synthesis. The activity of eEF1A has been shown by several studies to be regulated by post-translational modifications such as phosphorylation and dephosphorylation. However, until now less research has focused on other post-translational modifications of eEF1A, especially acetylation. In this report, we provide new evidence for the existence of eEF1A acetylation in PLC5 cells by immunoprecipitation and Western blotting. Using the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), we found that the deacetylation of eEF1A is mainly attributable to classes I and II HDAC rather than class III HDAC, and, furthermore, that the antitumour agent etoposide phosphate (VP 16) enhances the acetylation of eEF1A in a synergistic way with TSA. Our data suggest the possibility that the increased acetylation of eEF1A could be a new mechanism for the antitumour effect of etoposide.
Assuntos
Antineoplásicos/farmacologia , Etoposídeo/análogos & derivados , Compostos Organofosforados/farmacologia , Fator 1 de Elongação de Peptídeos/metabolismo , Acetilação , Linhagem Celular , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Etoposídeo/farmacologia , HumanosRESUMO
Caspases are a family of evolutionary conserved cysteine proteases that play key roles in programmed cell death and inflammation. Among the methods for the detection of caspase activity, biosensors based on luciferases have advantages in genetical encoding and convenience in assay. In this study, we constructed a new set of caspase biosensors based on NanoLuc luciferase. This kind of sensors, named NanoLock, work in dark-to-bright model, with the help of a NanoLuc quencher peptide (HiBiT-R/D) mutated from HiBiT. Optimized NanoLock responded to proteases with high signal to noise ratio (S/N), 1233-fold activation by tobacco etch virus protease in HEK293 cells and > 500-fold induction to caspase 3 in vitro. We constructed NanoLocks for the detection of caspase 1, 3, 6, 7, 8, 9, and 10, and assays in HEK293 cells demonstrated that these sensors performed better than commercial kits in the aspect of S/N and convenience. We further established a cell line stably expressing NanoLock-casp 6 and provided a proof-of-concept for the usage of this cell line in the high throughput screening of caspase 6 modulator.
Assuntos
Apoptose , Caspases , Caspase 3 , Caspases/genética , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismoRESUMO
One of the most desirable targets for HBV medications is the sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for the hepatitis B virus (HBV). N-myristoylated preS1 2-48 (Myrcludex B or Hepcludex), an NTCP-binding peptide from the large surface protein of HBV, has been developed as the first-in-class entry inhibitor. However, its relatively large molecular weight contributes to increased immunogenicity and antibody production. As a result, it is preferable to look for an NTCP-binding peptide with a smaller size. To do this, we developed a human cell surface display strategy and screened peptides based on preS1-21. PreS1-21 (genotype D) was extended by 7 random amino acids and fused with mCherry and FasL transmembrane domain. The pooled constructs were transfected into HEK293 cells by using the transposon/transposase system to create a library displaying various peptides on the cell surface with red fluorescence. On the other hand, we expressed NTCP protein fused with EGFP on HEK293 and used the membrane lysate containing NTCP-GFP as the bait protein to select peptides with increased NTCP affinity. After 7 cycles of selection, the deep sequencing results revealed that some polypeptides were more than 1,000 times enriched. Further screening of the mostly enriched 10 peptides yields the peptide preS1-21-pep3. Replacing the preS1-21 sequence of preS1-21-pep3 with those from different genotypes demonstrated that the consensus sequence of genotype A-F had the best performance. The peptide (Myr-preS1-21-pep3) was synthesized and tested on the HepG2-NTCP cell model. The results showed that Myr-preS1-21-pep3 is approximately 10 times more potent than the initial peptide Myr-preS1-21 in preventing HBV infection. In conclusion, we developed a new strategy for screening peptides binding to membrane proteins and identified a new NTCP-binding peptide with a much smaller size than Hepcludex.
RESUMO
Protein sensors based on allosteric enzymes responding to target binding with rapid changes in enzymatic activity are potential tools for homogeneous assays. However, a high signal-to-noise ratio (S/N) is difficult to achieve in their construction. A high S/N is critical to discriminate signals from the background, a phenomenon that might largely vary among serum samples from different individuals. Herein, based on the modularized luciferase NanoLuc, we designed a novel biosensor called NanoSwitch. This sensor allows direct detection of antibodies in 1 µl serum in 45 min without washing steps. In the detection of Flag and HA antibodies, NanoSwitches respond to antibodies with S/N ratios of 33-fold and 42-fold, respectively. Further, we constructed a NanoSwitch for detecting SARS-CoV-2-specific antibodies, which showed over 200-fold S/N in serum samples. High S/N was achieved by a new working model, combining the turn-off of the sensor with human serum albumin and turn-on with a specific antibody. Also, we constructed NanoSwitches for detecting antibodies against the core protein of hepatitis C virus (HCV) and gp41 of the human immunodeficiency virus (HIV). Interestingly, these sensors demonstrated a high S/N and good performance in the assays of clinical samples; this was partly attributed to the combination of off-and-on models. In summary, we provide a novel type of protein sensor and a working model that potentially guides new sensor design with better performance.
Assuntos
Técnicas Biossensoriais , COVID-19 , Anticorpos Antivirais , COVID-19/diagnóstico , Humanos , Luciferases , SARS-CoV-2RESUMO
The core promoter (CP) of hepatitis B virus (HBV) is critical for HBV replication by controlling the transcription of pregenomic RNA (pgRNA). Host factors regulating the activity of the CP can be identified by different methods. Biotin-based proximity labeling, a powerful method with the capability to capture weak or dynamic interactions, has not yet been used to map proteins interacting with the CP. Here, we established a strategy, based on the newly evolved promiscuous enzyme TurboID, for interrogating host factors regulating the activity of HBV CP. Using this strategy, we identified STAU1 as an important factor involved in the regulation of HBV CP. Mechanistically, STAU1 indirectly binds to CP mediated by TARDBP, and recruits the SAGA transcription coactivator complex to the CP to upregulate its activity. Moreover, STAU1 binds to HBx and enhances the level of HBx by stabilizing it in a ubiquitin-independent manner.