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      A genome-wide CRISPR screening uncovers that TOB1 acts as a key host factor for FMDV infection via both IFN and EGFR mediated pathways

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          Abstract

          The interaction between foot-and-mouth disease virus (FMDV) and the host is extremely important for virus infection, but there are few researches on it, which is not conducive to vaccine development and FMD control. In this study, we designed a porcine genome-scale CRISPR/Cas9 knockout library containing 93,859 single guide RNAs targeting 16,886 protein-coding genes, 25 long ncRNAs, and 463 microRNAs. Using this library, several previously unreported genes required for FMDV infection are highly enriched post-FMDV selection in IBRS-2 cells. Follow-up studies confirmed the dependency of FMDV on these genes, and we identified a functional role for one of the FMDV-related host genes: TOB1 (Transducer of ERBB2.1). TOB1-knockout significantly inhibits FMDV infection by positively regulating the expression of RIG-I and MDA5. We further found that TOB1-knockout led to more accumulation of mRNA transcripts of transcription factor CEBPA, and thus its protein, which further enhanced transcription of RIG-I and MDA5 genes. In addition, TOB1-knockout was shown to inhibit FMDV adsorption and internalization mediated by EGFR/ERBB2 pathway. Finally, the FMDV lethal challenge on TOB1-knockout mice confirmed that the deletion of TOB1 inhibited FMDV infection in vivo. These results identify TOB1 as a key host factor involved in FMDV infection in pigs.

          Author summary

          Host factors play a critical role in FMDV infection, however, the mechanisms are poorly understood. Aiming to comprehensively study the host factors involved in FMDV infection, we develop a porcine genome-scale CRISPR/Cas9 knockout library and successfully confirmed that the knockout of TOB1 (Transducer of ERBB2.1) could significantly inhibit FMDV infection. On one hand, TOB1-knockout could promote the expression of RIG-I and MDA5, and thus IFNB and ISGs (interferon stimulating genes), thereby inhibiting FMDV infection. On the other hand, TOB1-knockout inhibits FMDV entry mediated by EGFR/ERBB2 pathway. These findings reveal TOB1 as a key host factor mediating FMDV infection in pigs.

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          Most cited references48

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          Interferon-stimulated genes: a complex web of host defenses.

          Interferon-stimulated gene (ISG) products take on a number of diverse roles. Collectively, they are highly effective at resisting and controlling pathogens. In this review, we begin by introducing interferon (IFN) and the JAK-STAT signaling pathway to highlight features that impact ISG production. Next, we describe ways in which ISGs both enhance innate pathogen-sensing capabilities and negatively regulate signaling through the JAK-STAT pathway. Several ISGs that directly inhibit virus infection are described with an emphasis on those that impact early and late stages of the virus life cycle. Finally, we describe ongoing efforts to identify and characterize antiviral ISGs, and we provide a forward-looking perspective on the ISG landscape.
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            Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins

            Through the study of transcriptional activation in response to interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma), a previously unrecognized direct signal transduction pathway to the nucleus has been uncovered: IFN-receptor interaction at the cell surface leads to the activation of kinases of the Jak family that then phosphorylate substrate proteins called STATs (signal transducers and activators of transcription). The phosphorylated STAT proteins move to the nucleus, bind specific DNA elements, and direct transcription. Recognition of the molecules involved in the IFN-alpha and IFN-gamma pathway has led to discoveries that a number of STAT family members exist and that other polypeptide ligands also use the Jak-STAT molecules in signal transduction.
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              The JAK-STAT pathway at twenty.

              We look back on the discoveries that the tyrosine kinases TYK2 and JAK1 and the transcription factors STAT1, STAT2, and IRF9 are required for the cellular response to type I interferons. This initial description of the JAK-STAT pathway led quickly to additional discoveries that type II interferons and many other cytokines signal through similar mechanisms. This well-understood pathway now serves as a paradigm showing how information from protein-protein contacts at the cell surface can be conveyed directly to genes in the nucleus. We also review recent work on the STAT proteins showing the importance of several different posttranslational modifications, including serine phosphorylation, acetylation, methylation, and sumoylation. These remarkably proficient proteins also provide noncanonical functions in transcriptional regulation and they also function in mitochondrial respiration and chromatin organization in ways that may not involve transcription at all. Copyright © 2012 Elsevier Inc. All rights reserved.

                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: Methodology
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Methodology
                Role: Data curationRole: Formal analysisRole: Resources
                Role: Data curationRole: Formal analysis
                Role: Data curationRole: Formal analysis
                Role: Data curationRole: Formal analysis
                Role: Data curationRole: Formal analysis
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administration
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                PLOS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                21 March 2024
                March 2024
                : 20
                : 3
                : e1012104
                Affiliations
                [1 ] State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, National Engineering Laboratory for Animal Breeding, Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing, China
                [2 ] State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
                University of California, Irvine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0003-0764-6122
                https://orcid.org/0000-0003-4499-8610
                https://orcid.org/0000-0002-4831-5103
                https://orcid.org/0000-0002-1644-2300
                Article
                PPATHOGENS-D-23-01534
                10.1371/journal.ppat.1012104
                10986976
                38512977
                0b9cfd1c-bcb8-452d-b4b4-e6b9793b81a2
                © 2024 Peng et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 7 September 2023
                : 7 March 2024
                Page count
                Figures: 7, Tables: 2, Pages: 26
                Funding
                Funded by: National Key Research and Development Program of China
                Award ID: 2021YFD1800300
                Award Recipient :
                Funded by: National Key Research and Development Program of China
                Award ID: 2021YFA0805905
                Award Recipient :
                Funded by: National Key Research and Development Program of China
                Award ID: 2023YFF1000900
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100005236, Chinese Universities Scientific Fund;
                Award ID: 2022TC147
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 32272839
                Award Recipient :
                Funded by: Chinese Academy of Agricultural Science and Technology Innovation Project
                Award ID: CAAS-ASTIP-2020-LVRI
                Award Recipient :
                Funded by: Fundamental Research Funds of Chinese Academy of Agricultural Sciences
                Award ID: 1610312021010
                Award Recipient :
                Funded by: Fundamental Research Funds of Chinese Academy of Agricultural Sciences
                Award ID: 1610312021013
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100013909, State Key Laboratory of Veterinary Biotechnology;
                Award ID: SKLVEB2021DBCG01
                Award Recipient :
                This research was funded by the National Key Research and Development Program of China (2021YFD1800300 to DL, 2021YFA0805905 to SW, and 2023YFF1000900 to YZ), the Chinese Universities Scientific Fund (2022TC147 to YZ), the National Natural Science Foundation of China (32272839 to YZ), the Chinese Academy of Agricultural Science and Technology Innovation Project (CAAS-ASTIP-2020-LVRI to HZ), the Fundamental Research Funds of Chinese Academy of Agricultural Sciences (1610312021010 to HZ and 1610312021013 to HZ) and the State Key Laboratory of Veterinary Biotechnology (SKLVEB2021DBCG01 to HZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Zoology
                Animal Diseases
                Foot and Mouth Disease
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Transfection
                Research and Analysis Methods
                Molecular Biology Techniques
                Transfection
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Library Screening
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Library Screening
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                Small interfering RNA
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Small interfering RNA
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and life sciences
                Cell biology
                Signal transduction
                Cell signaling
                EGFR signaling
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Research and analysis methods
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Biology and Life Sciences
                Biochemistry
                Proteins
                Post-Translational Modification
                Phosphorylation
                Custom metadata
                vor-update-to-uncorrected-proof
                2024-04-02
                All relevant data are within the manuscript and its Supporting information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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