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: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: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: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:Interferon (IFN)-alpha causes high rates of depression and fatigue, and is used to investigate the impact of innate immune cytokines on brain and behavior. However, little is known about transcriptional profiles of circulating immune cells during chronic IFN-alpha administration. Accordingly, genome-wide transcriptional profiling was performed on peripheral blood mononuclear cells from 21 patients with chronic hepatitis C virus either awaiting IFN-alpha therapy (n=10) or after 12 weeks of IFN-alpha treatment (n=11). Significance analysis of microarray data identified 252 up-regulated gene transcripts, the majority of which were related to IFN-alpha/antiviral or innate-immune/inflammatory signaling. Of these upregulated genes, 2'-5'-oligoadenylate synthetase 2 (OAS2) was the only gene that was differentially expressed in patients that developed IFN-alpha-induced depression/fatigue, and correlated with depression and fatigue scores at 12 weeks of IFN-alpha administration. Promoter-based bioinformatic and cellular origin analyses revealed IFN-alpha-induced increases in genes bearing transcription factor binding motifs (TFBMs) related to myeloid differentiation, IFN-alpha signaling, API and CREB/ATF family of transcription pathways, with changes derived primarily from monocytes and plasmacytoid dendritic cells. Patients with high depression/fatigue scores demonstrated up-regulation of genes bearing TFBMs for myeloid differentiation, IFN-alpha and AP1 signaling, and down regulation of TFBMs for CREB/ATF-related transcription factors. Cellular origin analyses indicated a shift toward genes derived from CD8+T and NK cells in subjects with high depression/fatigue scores. These results reveal an antiviral and inflammatory transcriptional profile after 12 weeks IFN-alpha, accompanied by increased OAS2 expression, decreased CREB/ATF transcriptional control, and a shift from monocyte-derived genes to those of cytotoxic lymphocytes in IFN-alpha-induced depression/fatigue. Total RNA was isolated from the peripheral blood mononuclear cells (PBMC) obtained at 12 weeks from HCV patients treated with IFN-alpha plus ribavirin (n=11) and untreated HCV patients awaiting IFN-alpha/ribavirin therapy (control subjects, n=10).
2012-01-01 | E-GEOD-31187 | biostudies-arrayexpress
Project description:Viruses employ various strategies to evade innate immunity. Despite many studies on host or viral gene expression, how the cellular proteome responds to internal or external cues, has not been fully investigated. Using a Hepatitis B Virus (HBV) replication model, we performed proteomic analyses of HBV-replicating cells in G1/S and G2/M phases, as a function of IFN-alpha, providing specific information of how HBV infection, in combination with IFN-alpha, alters the hepatocyte proteome. We identified the conserved LSm (Like-Sm1-8) proteins were differentially expressed as a function of HBV replication and IFN-alpha. Specifically, in G2/M, IFN-alpha increased protein level of LSm1, the unique subunit of cytoplasmic LSm1-7 complex involved in mRNA decay. By contrast, IFN-alpha decreased LSm8, the unique subunit of nuclear LSm2-8 complex, a chaperone of U6 spliceosomal RNA. These results suggest cytoplasmic LSm1-7 has antiviral role, whereas nuclear LSm2-8 complex is pro-viral. Employing HBV replication and infection models, siRNA-mediated knockdown of LSm1 increased the level of all viral RNAs. Conversely, knockdown of LSm8 reduced viral RNA levels, dependent on N6-adenosine methylation (m6A) of the epsilon stem-loop at the 5 end of pre-Core/pregenomic (preC/pg) RNA. Methylated RNA immunoprecipitation (MeRIP) assays demonstrated reduced viral RNA methylation upon LSm8 knockdown, dependent on the m6A modification, suggesting the LSm2-8 complex has a role in mediating this modification. Interestingly, splicing inhibitor Cp028 acting upstream of LSm2-8 complex, suppressed levels of viral RNAs without reducing the m6A modification. This observation suggests Cp028 has novel antiviral effects, likely potentiating IFN-alpha -mediated suppression of HBV biosynthesis.
2022-06-03 | MSV000089600 | MassIVE