Interferon antagonism by SARS-CoV-2: a functional study using reverse genetics
Summary: Background: The COVID-19 agent, SARS-CoV-2, is conspecific with SARS-CoV, the causal agent of the severe acute respiratory syndrome epidemic in 2002–03. Although the viruses share a completely homologous repertoire of proteins and use the same cellular entry receptor, their transmission ef...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-05-01
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| Series: | The Lancet Microbe |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666524721000276 |
| _version_ | 1818642562893742080 |
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| author | Simon Schroeder, MSc Fabian Pott, MSc Daniela Niemeyer, PhD Talitha Veith, MPhil Anja Richter Doreen Muth, PhD Christine Goffinet, ProfPhD Marcel A Müller, PhD Christian Drosten, ProfPhD |
| author_facet | Simon Schroeder, MSc Fabian Pott, MSc Daniela Niemeyer, PhD Talitha Veith, MPhil Anja Richter Doreen Muth, PhD Christine Goffinet, ProfPhD Marcel A Müller, PhD Christian Drosten, ProfPhD |
| author_sort | Simon Schroeder, MSc |
| collection | DOAJ |
| description | Summary: Background: The COVID-19 agent, SARS-CoV-2, is conspecific with SARS-CoV, the causal agent of the severe acute respiratory syndrome epidemic in 2002–03. Although the viruses share a completely homologous repertoire of proteins and use the same cellular entry receptor, their transmission efficiencies and pathogenetic traits differ. We aimed to compare interferon antagonism by SARS-CoV and SARS-CoV-2. Methods: For this functional study, we infected Vero E6 and Calu-3 cells with strains of SARS-CoV and SARS-CoV-2. We studied differences in cell line-specific replication (Vero E6 vs Calu-3 cells) and analysed these differences in relation to TMPRSS2-dependent cell entry based on inhibition with the drug camostat mesilate. We evaluated viral sensitivity towards type I interferon treatment and assessed cytokine induction and type I interferon signalling in the host cells by RT-PCR and analysis of transcription factor activation and nuclear translocation. Based on reverse genetic engineering of SARS-CoV, we investigated the contribution of open reading frame 6 (ORF6) to the observed phenotypic differences in interferon signalling, because ORF6 encodes an interferon signalling antagonist. We did a luciferase-based interferon-stimulated response element promotor activation assay to evaluate the antagonistic capacity of SARS-CoV-2 wild-type ORF6 constructs and three mutants (Gln51Glu, Gln56Glu, or both) that represent amino acid substitutions between SARS-CoV and SARS-CoV-2 protein 6 in the carboxy-terminal domain. Findings: Overall, replication was higher for SARS-CoV in Vero E6 cells and for SARS-CoV-2 in Calu-3 cells. SARS-CoV-2 was reliant on TMPRSS2, found only in Calu-3 cells, for more efficient entry. SARS-CoV-2 was more sensitive to interferon treatment, less efficient in suppressing cytokine induction via IRF3 nuclear translocation, and permissive of a higher level of induction of interferon-stimulated genes MX1 and ISG56. SARS-CoV-2 ORF6 expressed in the context of a fully replicating SARS-CoV backbone suppressed MX1 gene induction, but this suppression was less efficient than that by SARS-CoV ORF6. Mutagenesis showed that charged amino acids in residues 51 and 56 shift the phenotype towards more efficient interferon antagonism, as seen in SARS-CoV. Interpretation: SARS-CoV-2 ORF6 interferes less efficiently with human interferon induction and interferon signalling than SARS-CoV ORF6. Because of the homology of the genes, onward selection for fitness could involve functional optimisation of interferon antagonism. Charged amino acids at positions 51 and 56 in ORF6 should be monitored for potential adaptive changes. Funding: Bundesministerium für Bildung und Forschung, EU RECOVER project. |
| first_indexed | 2024-12-16T23:45:02Z |
| format | Article |
| id | doaj.art-819ce5aee3df4748b4a4a8a997aedc0c |
| institution | Directory Open Access Journal |
| issn | 2666-5247 |
| language | English |
| last_indexed | 2024-12-16T23:45:02Z |
| publishDate | 2021-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | The Lancet Microbe |
| spelling | doaj.art-819ce5aee3df4748b4a4a8a997aedc0c2022-12-21T22:11:31ZengElsevierThe Lancet Microbe2666-52472021-05-0125e210e218Interferon antagonism by SARS-CoV-2: a functional study using reverse geneticsSimon Schroeder, MSc0Fabian Pott, MSc1Daniela Niemeyer, PhD2Talitha Veith, MPhil3Anja Richter4Doreen Muth, PhD5Christine Goffinet, ProfPhD6Marcel A Müller, PhD7Christian Drosten, ProfPhD8Institute of Virology, Charité-Universitätsmedizin Berlin, Berlin, GermanyInstitute of Virology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Berlin Institute of Health, Berlin, GermanyInstitute of Virology, Charité-Universitätsmedizin Berlin, Berlin, GermanyInstitute of Virology, Charité-Universitätsmedizin Berlin, Berlin, GermanyInstitute of Virology, Charité-Universitätsmedizin Berlin, Berlin, GermanyInstitute of Virology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Deutsches Zentrum für Infektionsforschung, Associated Partner Charité-Universitätsmedizin Berlin, Berlin, GermanyInstitute of Virology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Berlin Institute of Health, Berlin, GermanyInstitute of Virology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov University, Moscow, RussiaInstitute of Virology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Berlin Institute of Health, Berlin, Germany; Deutsches Zentrum für Infektionsforschung, Associated Partner Charité-Universitätsmedizin Berlin, Berlin, Germany; Correspondence to: Prof Christian Drosten, Institute of Virology, Charité-Universitätsmedizin, Berlin 10117, GermanySummary: Background: The COVID-19 agent, SARS-CoV-2, is conspecific with SARS-CoV, the causal agent of the severe acute respiratory syndrome epidemic in 2002–03. Although the viruses share a completely homologous repertoire of proteins and use the same cellular entry receptor, their transmission efficiencies and pathogenetic traits differ. We aimed to compare interferon antagonism by SARS-CoV and SARS-CoV-2. Methods: For this functional study, we infected Vero E6 and Calu-3 cells with strains of SARS-CoV and SARS-CoV-2. We studied differences in cell line-specific replication (Vero E6 vs Calu-3 cells) and analysed these differences in relation to TMPRSS2-dependent cell entry based on inhibition with the drug camostat mesilate. We evaluated viral sensitivity towards type I interferon treatment and assessed cytokine induction and type I interferon signalling in the host cells by RT-PCR and analysis of transcription factor activation and nuclear translocation. Based on reverse genetic engineering of SARS-CoV, we investigated the contribution of open reading frame 6 (ORF6) to the observed phenotypic differences in interferon signalling, because ORF6 encodes an interferon signalling antagonist. We did a luciferase-based interferon-stimulated response element promotor activation assay to evaluate the antagonistic capacity of SARS-CoV-2 wild-type ORF6 constructs and three mutants (Gln51Glu, Gln56Glu, or both) that represent amino acid substitutions between SARS-CoV and SARS-CoV-2 protein 6 in the carboxy-terminal domain. Findings: Overall, replication was higher for SARS-CoV in Vero E6 cells and for SARS-CoV-2 in Calu-3 cells. SARS-CoV-2 was reliant on TMPRSS2, found only in Calu-3 cells, for more efficient entry. SARS-CoV-2 was more sensitive to interferon treatment, less efficient in suppressing cytokine induction via IRF3 nuclear translocation, and permissive of a higher level of induction of interferon-stimulated genes MX1 and ISG56. SARS-CoV-2 ORF6 expressed in the context of a fully replicating SARS-CoV backbone suppressed MX1 gene induction, but this suppression was less efficient than that by SARS-CoV ORF6. Mutagenesis showed that charged amino acids in residues 51 and 56 shift the phenotype towards more efficient interferon antagonism, as seen in SARS-CoV. Interpretation: SARS-CoV-2 ORF6 interferes less efficiently with human interferon induction and interferon signalling than SARS-CoV ORF6. Because of the homology of the genes, onward selection for fitness could involve functional optimisation of interferon antagonism. Charged amino acids at positions 51 and 56 in ORF6 should be monitored for potential adaptive changes. Funding: Bundesministerium für Bildung und Forschung, EU RECOVER project.http://www.sciencedirect.com/science/article/pii/S2666524721000276 |
| spellingShingle | Simon Schroeder, MSc Fabian Pott, MSc Daniela Niemeyer, PhD Talitha Veith, MPhil Anja Richter Doreen Muth, PhD Christine Goffinet, ProfPhD Marcel A Müller, PhD Christian Drosten, ProfPhD Interferon antagonism by SARS-CoV-2: a functional study using reverse genetics The Lancet Microbe |
| title | Interferon antagonism by SARS-CoV-2: a functional study using reverse genetics |
| title_full | Interferon antagonism by SARS-CoV-2: a functional study using reverse genetics |
| title_fullStr | Interferon antagonism by SARS-CoV-2: a functional study using reverse genetics |
| title_full_unstemmed | Interferon antagonism by SARS-CoV-2: a functional study using reverse genetics |
| title_short | Interferon antagonism by SARS-CoV-2: a functional study using reverse genetics |
| title_sort | interferon antagonism by sars cov 2 a functional study using reverse genetics |
| url | http://www.sciencedirect.com/science/article/pii/S2666524721000276 |
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