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      A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

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          Abstract

          Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged—Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)—demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

          Abstract

          This study identifies essential host dependency factors for human coronavirus replication, showing that these can be directly targeted by clinically approved inhibitors and that treatment leads to effective inhibition of coronavirus replication in primary human nasal epithelial cell cultures.

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          Fiji: an open-source platform for biological-image analysis.

          Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
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            SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

            Summary The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
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              Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

              Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draft
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: Formal analysisRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Writing – review & editing
                Role: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: Writing – review & editing
                Role: Data curationRole: Investigation
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: MethodologyRole: Supervision
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: MethodologyRole: Supervision
                Role: ConceptualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: SupervisionRole: Writing – review & editing
                Role: Data curationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Academic Editor
                Journal
                PLoS Biol
                PLoS Biol
                plos
                PLoS Biology
                Public Library of Science (San Francisco, CA USA )
                1544-9173
                1545-7885
                28 December 2021
                December 2021
                28 December 2021
                : 19
                : 12
                : e3001490
                Affiliations
                [1 ] Institute of Virology and Immunology, Bern and Mittelhäusern, Switzerland
                [2 ] Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland
                [3 ] Graduate School for Biomedical Science, University of Bern, Bern, Switzerland
                [4 ] European Virus Bioinformatics Center, Jena, Germany
                [5 ] Department for Molecular & Medical Virology, Ruhr-Universität Bochum, Germany
                [6 ] Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Bern, Switzerland
                [7 ] Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz-Center Munich, Member of the German Center of Lung Research (DZL), Munich, Germany
                [8 ] Max von Pettenkofer-Institute, Ludwig-Maximilians-Universität München, Munich, Germany
                [9 ] German Center for Infection Research, Munich Site, Munich, Germany
                [10 ] Institute for Infectious Diseases, University of Bern, Bern, Switzerland
                New York University School of Medicine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                ‡ Equally shared senior authorship on this work.

                Author information
                https://orcid.org/0000-0001-8468-4742
                https://orcid.org/0000-0001-7265-2233
                https://orcid.org/0000-0002-8366-1220
                https://orcid.org/0000-0002-3564-1014
                https://orcid.org/0000-0002-1211-7834
                https://orcid.org/0000-0003-0320-2743
                https://orcid.org/0000-0002-2708-2507
                https://orcid.org/0000-0002-3957-5448
                Article
                PBIOLOGY-D-21-00778
                10.1371/journal.pbio.3001490
                8741300
                34962926
                7180c92f-ee01-4930-89d5-27216a5630d7
                © 2021 Kratzel 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
                : 19 March 2021
                : 22 November 2021
                Page count
                Figures: 5, Tables: 6, Pages: 35
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001711, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung;
                Award ID: 165076
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001711, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung;
                Award ID: 173085
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100010665, H2020 Marie Skłodowska-Curie Actions;
                Award ID: 748627
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002347, Bundesministerium für Bildung und Forschung;
                Award ID: 01KI1723A
                Award Recipient :
                This study was supported by the Swiss National Science Foundation (grant number 165076 and 173085, VT). In addition this study was funded by the Horizon 2020 Marie Skłodowska-Curie "COV RESTRICT" grant agreement (grant number 748627, SP) and the Federal Ministry of Education and Research (RAPID grant number 01KI1723A, VT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                All relevant data are within the paper and its Supporting Information files. Raw data from the CRISPR screens is deposited on the NCBI Sequence Read Archive (SRA) public repository (BioProject ID: PRJNA779941).
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