Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has caused a global health emergency. A key feature of COVID-19 is dysregulated Interferon-response. Type-I interferon (IFN-I) is one of the earliest antiviral innate immune responses following viral infection and plays a significant role in pathogenesis of SARS-CoV-2. In this study, using a proteomics-based approach, we identified that SARS-CoV-2 infection induces delayed and dysregulated IFN-I signaling in Huh7 cells. We demonstrate that SARS-CoV-2 is able to inhibit RIG-I mediated IFN- production. Our results also confirm the recent findings that IFN-I pretreatment is able to reduce susceptibility of Huh7 cells to SARS-CoV-2, but not post-treatment. Senescent Huh7 cells in spite of showing accentuated IFN-I response were more susceptible to SARS-CoV-2 infection and SARS-CoV-2 effectively inhibited IFIT1 in these cells. Proteomic comparison between SARS-CoV-2, SARS-CoV and MERS-CoV revealed a distinct differential regulatory signature of interferon-related proteins emphasizing that therapeutic strategies based on observations in SARS-CoV and MERS-CoV should be used with caution. Our findings provide a better understanding of SARS-CoV-2 regulation of cellular interferon response and a perspective on its use as a treatment. Characterization of the role of different interferon stimulated genes on the inhibition of SARS-CoV-2 pathogenesis may direct novel antiviral strategies.
Project description:The transcriptome of Huh7 and Huh7.5 was sequenced from cells treated with interferon alpha (IFNα), beta (IFNβ) or lambda (IFNλ) for 8 hours. Each treatment was compared to respective mock-treated cells in 6 pairwise comparisons, for a total of 43 samples classified in 12 experimental conditions. Cells were treated for 8h with 10ng/ml of Interferon-α2A, 5000 U/ml of Interferon-β1A or 100ng/ml of interferon-λ3 prior RNA extraction and library preparation. Mock-treated cells were used as control for each treatment. 3 to 4 biological replicates were analysed for each biological condition. Libraries for Illumina sequencing were constructed by polyA selection with the Illumina Kit. A paired end (2x100) run was performed.
Project description:The RNA modification N6-methyladenosine (m6A) regulates gene expression through various transcript-specific effects. The overall goal of these experiments was to determine the effects of the m6A methyltransferase complex proteins METTL3 and METTL14 on the expression of interferon stimulated genes. First, we depleted METTL3 and METTL14 in Huh7 cells using siRNAs and treated with Mock or IFN-β to analyze the regulatory effects of METTL3/14 on interferon-stimulated genes' transcript abundance. We then used Ribo-seq following METTL3/14 siRNA depletion and IFN treatment to quantify its effect on the translation of ISGs and other genes. Overall, we found that METTL3/14 exerts transcript-specific regulatory effects on a subset of interferon-stimulated genes. Overall, METTL3/14 appeared to enhance the translation of a subset of interferon-stimulated genes. We characterized the molecular effects of METTL3/14 and m6A on certain ISGs and determined that m6A augments the antiviral effects of the interferon response.
Project description:To understand the roles of JMJD5 in liver cells, we performed DNA microarray analysis by using JMJD5KO Huh7 cells. We noticed that several transcriptional factors involved in differentiation to hepatocytes were down-regulated in JMJD5KO Huh7 cells compared to parent Huh7 cells..
Project description:A powerful approach to study innate antiviral response is to compare the difference between wild type Huh7 cells, which do not support robust replication of hepatitis C virus (HCV)2, versus certain subclones of Huh7 cells that are permissive for HCV replication. We generated two permissive cell lines and two independent non-permissive subclone from Huh7 cells. We compared the global methylation pattern of these different cells and find that Huh7 cells exist as a heterogeneous population of cells with distinct patterns of gene methylation. Comparison of Huh7, HRP1, HRP4, Huh7-pNeo1 and Huh7-pNeo2 cells.
Project description:All major types of interferon (IFN) efficiently inhibit hepatitis C virus (HCV) replication in vitro and in vivo. Remarkably, HCV replication is not sensitive to IFN? in the hepatoma cell line Huh6, despite an intact signaling pathway. We performed transcriptome analyses between Huh6 and Huh-7 to identify effector genes of the IFN? response and thereby identified the DExD/H box helicase DDX60L as a restriction factor of HCV replication. DDX60L and its homolog DDX60 were both induced upon viral infection and IFN treatment in primary human hepatocytes. However, exclusively DDX60L knockdown increased HCV replication in Huh-7 cells, and rescued HCV replication from type II IFN as well as type I and III IFN treatment, suggesting that DDX60L is an important effector protein of the innate immune response against HCV. DDX60L had no impact on replication of hepatitis A virus (HAV), but severely impaired production of lentiviral vectors, arguing for a potential antiretroviral activity. Detection of endogenous DDX60L protein turned out to be difficult due to instability. DDX60L knockdown did not alter interferon stimulated gene (ISG) induction after IFN treatment, suggesting that it is a direct effector of the innate immune response. It most likely inhibits viral RNA replication, since we found no impact of DDX60L on translation or stability of HCV subgenomic replicons, nor additional impact on entry and assembly of infectious virus. Similar to its homolog DDX60, DDX60L had a moderate impact on retinoic acid-inducible gene I (RIG-I)-dependent activation of innate immunity arguing for additional functions in the sensing of viral RNA. Gene Expression was compared between two cell lines, Huh6 and Huh7, under interferon-gamma or interferon-alpha treatment. We intended to identify genes that are more strongly upregulated in Huh-7 than in Huh6 in response to interferon treatment.
Project description:The change of gene expression in Huh7 cells treated with IFN-λ4, IFN-λ1 and IFN-α was analyzed at 0h, 6h and 16h after different interferon treatments. Huh7 cells that had been treated with IFN-α (10 IU/ml), IFN-λ1 (20 ng/ml) and IFN-λ4 (20 ng/ml). Time points of 6 h and 16 h after stimulation were selected.