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A simple and robust cell culture system is essential for generating authentic SARS-CoV-2 stocks for evaluation of viral pathogenicity, screening of antiviral compounds, and preparation of inactivated vaccines. Evidence suggests that Vero E6, a cell line commonly used in the field to grow SARS-CoV-2, does not support efficient propagation of new viral variants and triggers rapid cell culture adaptation of the virus. We generated a panel of 17 human cell lines overexpressing SARS-CoV-2 entry factors and tested their ability to support viral infection. Two cell lines, Caco-2/AT and HuH-6/AT, demonstrated exceptional susceptibility, yielding highly concentrated virus stocks. Notably, these cell lines were more sensitive than Vero E6 cells in recovering SARS-CoV-2 from clinical specimens. Further, Caco-2/AT cells provided a robust platform for producing genetically reliable recombinant SARS-CoV-2 through a reverse genetics system. These cellular models are a valuable tool for the study of SARS-CoV-2 and its continuously emerging variants.
RESUMO
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than 235 million cases worldwide and 4.8 million deaths (October 2021), with various incidences and mortalities among regions/ethnicities. The coronaviruses SARS-CoV, SARS-CoV-2, and HCoV-NL63 utilize the angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2. We then applied an integrated approach of genetics, biochemistry, and virology to explore the capacity of select ACE2 variants to bind coronavirus spike proteins and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity. IMPORTANCE There is considerable variation in disease severity among patients infected with SARS-CoV-2, the virus that causes COVID-19. Human genetic variation can affect disease outcome, and the coronaviruses SARS-CoV, SARS-CoV-2, and HCoV-NL63 utilize human ACE2 as the receptor to enter cells. We found that several missense ACE2 single-nucleotide variants (SNVs) that showed significantly altered binding with the spike proteins of SARS-CoV, SARS-CoV-2, and NL63-HCoV. We identified an ACE2 SNP, D355N, that restricts the spike protein-ACE2 interaction and consequently has the potential to protect individuals against SARS-CoV-2 infection. Our study highlights that ACE2 polymorphisms could impact human susceptibility to SARS-CoV-2, which may contribute to ethnic and geographical differences in SARS-CoV-2 spread and pathogenicity.
Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Predisposição Genética para Doença/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Variação Genética , Humanos , Polimorfismo de Nucleotídeo Único , Ligação Proteica , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do VírusRESUMO
SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.
Assuntos
COVID-19/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Janus Quinases/metabolismo , SARS-CoV-2/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Evasão da Resposta Imune , Imunidade Inata , Interferon Tipo I/metabolismo , Janus Quinase 1/metabolismo , Miócitos Cardíacos , Receptor de Interferon alfa e beta/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , TYK2 Quinase/metabolismo , Replicação ViralRESUMO
The need to stem the current outbreak of SARS-CoV-2 responsible for COVID-19 is driving the search for inhibitors that will block coronavirus replication and pathogenesis. The coronavirus 3C-like protease (3CLpro) encoded in the replicase polyprotein is an attractive target for antiviral drug development because protease activity is required for generating a functional replication complex. Reagents that can be used to screen for protease inhibitors and for identifying the replicase products of SARS-CoV-2 are urgently needed. Here we describe a luminescence-based biosensor assay for evaluating small molecule inhibitors of SARS-CoV-2 3CLpro/main protease. We also document that a polyclonal rabbit antiserum developed against SARS-CoV 3CLpro cross reacts with the highly conserved 3CLpro of SARS-CoV-2. These reagents will facilitate the pre-clinical evaluation of SARS-CoV-2 protease inhibitors.
Assuntos
Técnicas Biossensoriais/métodos , Proteases 3C de Coronavírus/metabolismo , Soros Imunes/imunologia , Luciferases/metabolismo , SARS-CoV-2/metabolismo , Animais , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/imunologia , Reações Cruzadas , Luciferases/genética , Inibidores de Proteases/farmacologia , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.
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COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Imunoglobulina A/imunologia , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Chlorocebus aethiops , Reações Cruzadas , Epitopos , Células HEK293 , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina A Secretora/imunologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células VeroRESUMO
COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity or as a therapeutic has yet been developed to SARS-CoV-2. In this study we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks hACE2 receptor binding, by completely overlapping the hACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in human epithelial cells expressing hACE2. SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.