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SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

ABSTRACT: The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

SUBMITTER: Hou YJ

PROVIDER: S-EPMC7250779 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

Hou Yixuan J YJ Okuda Kenichi K Edwards Caitlin E CE Martinez David R DR Asakura Takanori T Dinnon Kenneth H KH Kato Takafumi T Lee Rhianna E RE Yount Boyd L BL Mascenik Teresa M TM Chen Gang G Olivier Kenneth N KN Ghio Andrew A Tse Longping V LV Leist Sarah R SR Gralinski Lisa E LE Schäfer Alexandra A Dang Hong H Gilmore Rodney R Nakano Satoko S Sun Ling L Fulcher M Leslie ML Livraghi-Butrico Alessandra A Nicely Nathan I NI Cameron Mark M Cameron Cheryl C Kelvin David J DJ de Silva Aravinda A Margolis David M DM Markmann Alena A Bartelt Luther L Zumwalt Ross R Martinez Fernando J FJ Salvatore Steven P SP Borczuk Alain A Tata Purushothama R PR Sontake Vishwaraj V Kimple Adam A Jaspers Ilona I O'Neal Wanda K WK Randell Scott H SH Boucher Richard C RC Baric Ralph S RS

Cell 20200527 2

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting ...[more]

PMID: 32526206

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Project description:BackgroundThe 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.MethodsFor 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.FindingsOverall, 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.InterpretationSARS-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.FundingBundesministerium für Bildung und Forschung, EU RECOVER project.

Dataset Information

SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

Publications

SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

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