Interferon Alpha Induces Multiple Cellular Proteins That Coordinately Suppress Hepadnaviral Covalently Closed Circular DNA Transcription
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ABSTRACT:
ORGANISM(S): Gallus gallus
PROVIDER: GSE134325 | GEO | 2020/06/17
REPOSITORIES: GEO
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Project description:Hepatitis B virus (HBV) contains a partially double-stranded relaxed circular DNA (rcDNA) genome that is converted into a covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocyte by cellular DNA repair machinery. cccDNA associates with nucleosomes to form a minichromosome that transcribes RNA to support the expression of viral proteins and reverse transcriptional replication of viral DNA. In addition to the de novo synthesis from incoming virion rcDNA, cccDNA can also been synthesized from rcDNA in the progeny nucleocapsids in the cytoplasm of infected hepatocytes via the intracellular amplification pathway. In our efforts to identify cellular DNA repair proteins required for cccDNA synthesis by a chemogenetic screen, we found that B02, a small molecular inhibitor of DNA homologous recombination repair protein RAD51, significantly enhanced the synthesis of cccDNA via intracellular amplification pathway in human hepatoma cells. Ironically, neither siRNA knockdown of RAD51 expression nor treatment with another structurally distinct RAD51 inhibitor or activator altered cccDNA amplification. However, further mechanistic studies revealed that B02 treatment significantly elevated the levels of multiple heat shock protein mRNA and siRNA knockdown of HSPA1A expression or treatment with HSPA1 inhibitors significantly attenuated B02 enhancement of cccDNA amplification. Moreover, B02 enhanced cccDNA amplification was efficiently inhibited by compounds that selectively inhibit DNA polymerase α or topoisomerase II, the enzymes required for cccDNA intracellular amplification. Our results thus indicate that B02 treatment induces a heat shock protein-mediated cellular response that positively regulates the conversion of rcDNA into cccDNA via the authentic intracellular amplification pathway
2022-12-08 | GSE209863 | GEO
Project description:Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.
| S-EPMC7431811 | biostudies-literature
Project description:Objective: Curing hepatitis B requires the complete elimination of covalently closed circular DNA (cccDNA). Interferon (IFN)-γ is produced by cytotoxic T lymphocytes and has noncytolytic antiviral potential; however, elimination of cccDNA could not be achieved. To enhance the regulatory effect of IFN-γ, we comprehensively analyzed the host factors that associated with cccDNA amplification and IFN-γ effects using the in vitro HBV infection system that exhibits various transcription levels. Design: Primary human hepatocytes were infected with HBV using genomic plasmids carrying the basic core promoter 1762/1764 and/or the precore 1896 mutation and treated with IFN-γ, IFN-α, and entecavir. Comprehensive expression analysis and functional studies were performed to analyze the host factors related to the cccDNA regulation using RNA microarray and siRNA analysis. Results: HBV infection system accurately reproduced the HBV life cycle and exhibited various transcription levels. Microarray analysis revealed that 53 genes increased depending on the cccDNA levels. Of 53 genes, the expression of IFN-induced protein 44-like (IFI44L) was the most upregulated by IFN-γ and IFN-α but not entecavir, and associated with the anti-viral effects of IFN-γ. siRNA analysis revealed that IFI44L negatively regulates the innate immune response and IFN-γ function to suppress HBV transcription and propagation by inhibiting the activation of NF-κB and STAT1 pathways.
2020-12-12 | GSE163042 | GEO
Project description:Interferon Alpha Induces Multiple Cellular Proteins That Coordinately Suppress Hepadnaviral Covalently Closed Circular DNA Transcription
| PRJNA554783 | ENA
Project description:Hepatitis B virus (HBV) infection is a risk of developing fibrosis, cirrhosis, liver failure, and hepatocellular carcinoma. Although HBV elimination requires complete elimination of covalently closed circular DNA (cccDNA), its treatment has not been established. Interferon (IFN) -γ, a type ⅠⅠ IFN, is produced by intrahepatic cytotoxic T lymphocytes and has the noncytolytic antiviral potential. However, the mechanism by which IFN-γ regulates HBV infection in hepatocytes has not been fully elucidated. In this study, to replicate the HBV infection and monitor the amount of cccDNA, we developed an in vitro HBV infection assay system with primary hepatocytes and examined the molecules and signaling pathways. IFN-γ suppressed both HBV propagation and transcription to the same extent as IFN-α. RNA microarray analysis revealed that IFN-γ stimulation induced not only IFN-γ but also IFN-α signaling activation and regulated HBV cccDNA. Moreover, the HBV production was reduced by IFN-γ through JAK-STAT signaling and interferon stimulated genes such as OAS2 and APOBEC3G. Taken together, these results demonstrate that IFN-γ suppresses both HBV propagation and transcription by activating specific intracellular signaling pathways in hepatocytes and suggests the future application of this particular signaling pathways or genes for the complete elimination of HBV.
2019-10-01 | GSE132628 | GEO
Project description:Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies and have been successfully employed for the treatment of viral diseases. Humans express twelve IFN-alpha (α) subtypes, which activate downstream signalling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in type I IFN immunity and the presence of anti- IFN autoantibodies account for very severe courses of COVID-19, therefore, early administration of type I IFNs may be protective against life-threatening disease. Here we comprehensively analysed the antiviral activity of all IFNα subtypes against SARS-CoV-2 to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate and low antiviral IFNs. In particular IFNα5 showed superior antiviral activity against SARS-CoV-2 infection. Dose-dependency studies further displayed additive effects upon co-administered with the broad antiviral drug remdesivir in cell culture. Transcriptomics of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in type I IFN signalling pathways, negative regulation of viral processes and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multi-modular definition of antiviral host responses mediated by defined type I IFNs. This knowledge shall support the development of novel therapeutic approaches against SARS-CoV-2.
2022-02-09 | PXD026079 | Pride
Project description:Background & Aims: IFN-alpha and -gamma have been reported to suppress cccDNA via APOBEC3 cytidine deaminase activity, and CDM-3008, an IFNAR2 agonist, also suppress cccDNA through interferon signaling. In order to develop a novel anti-HBV drug for functional or complete cure, we performed in silico screening using a structure of CDM-3008-binding pocket in IFNAR2. Approach & Results: The binding pocket of CDM-3008 in IFNAR2 was determined by LC-MS/MS using a CDM-3008-based molecular probe, CDM-3095 and a recombinant protein of IFNAR2 extracellular region. We conducted in silico screening of the binding compounds fitting the steric structure of the pocket. We identified 37 compounds and named them iCDM-1–37. We found that iCDM-34 showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 with or without entecavir (ETV). iCDM-34 suppressed pregenome RNA, covalently closed circular DNA (cccDNA), HBsAg, and HBeAg and clearly exhibited synergistic inhibitory effects with ETV. Metabolic stability of iCDM-34 was measured in human, mouse, and rat microsomal fractions. iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single agent, but when combined with ETV, it suppressed HBV DNA and cccDNA compared to ETV alone. Phosphoproteome analysis showed no activation of IFN signaling with iCDM-34 treatment. Transcriptome analysis of interferon-stimulated genes (ISGs) revealed no increase in the expression, while downstream factors of aryl hydrocarbon receptor (AhR) showed increased expression. Conclusion: iCDM-34 is a novel anti-HBV drug that induces AhR activation and suppresses cccDNA. Conclusion: iCDM-34 is a novel anti-HBV drug that induces AhR activation and suppresses cccDNA.
2022-03-25 | GSE180646 | GEO
Project description:mRNA expression profiles in primary microglia cells treated with R848, IFN-α, and R848+ IFN-α
| PRJNA408123 | ENA
Project description:: Hepatitis B virus (HBV) remains a major public health threat with more than 296 million people chronically infected worldwide at high risk to develop hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. The fundamental limitation rests on the fact that current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. HBV core protein (HBc) is associated with cccDNA. We used an anti-HBc antibody to pull down HBcAg-associated cccDNA complex in nuclear extract of HBV-infected HepG2-NTCP cells. By protein mass spectrometry, we identified many host proteins, including previously known proteins, that are associated with HBV cccDNA. Some of these proteins were functionally validated to play a role in HBV cccDNA activities.
2024-06-16 | PXD042733 | Pride
Project description:IFN alpha mediated gene expression pattern. The effect of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. This analysis examined the effects of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. Magnetically sorted untouched CD8+CD45R0- T cells from three different donors were unstimulated or stimulated with IFNa2b or with anti-CD3/CD28 Beads alone or along with IFNa2b or IFNa5 for 48 hours. Individual mRNA samples were analyzed using HG-U133A 2.0 array gene chips. Keywords: Gene expression analysis after different stimulation Magnetically sorted untouched CD8+CD45R0-T cells from three different donors (named A, B and C) unstimulated or stimulated with IFN alpha 2b or with anti-CD3/CD28 beads alone or along with IFN alpha 2b or IFN alpha 5 for 48 hours.
2010-07-23 | E-GEOD-17301 | biostudies-arrayexpress