RESUMO
The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.
Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/metabolismo , Proteômica/métodos , Células A549 , Enzima de Conversão de Angiotensina 2 , Animais , Antivirais/farmacologia , COVID-19 , Células CACO-2 , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosforilação , Pneumonia Viral/virologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Receptor Tirosina Quinase AxlRESUMO
We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.
Assuntos
Epistasia Genética , Infecções por HIV/genética , Fator Regulador 7 de Interferon/genética , Fatores de Transcrição/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Interferons/genética , Mutação , Transdução de Sinais/genéticaRESUMO
A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.
Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Reposicionamento de Medicamentos , Terapia de Alvo Molecular , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Mapas de Interação de Proteínas , Proteínas Virais/metabolismo , Animais , Antivirais/classificação , Antivirais/farmacologia , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , COVID-19 , Chlorocebus aethiops , Clonagem Molecular , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Imunidade Inata , Espectrometria de Massas , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Domínios Proteicos , Mapeamento de Interação de Proteínas , Receptores sigma/metabolismo , SARS-CoV-2 , Proteínas Ligases SKP Culina F-Box/metabolismo , Células Vero , Proteínas Virais/genética , Tratamento Farmacológico da COVID-19RESUMO
The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.
Assuntos
COVID-19/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Interações entre Hospedeiro e Microrganismos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mapas de Interação de Proteínas , SARS-CoV-2/metabolismo , Síndrome Respiratória Aguda Grave/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Sequência Conservada , Proteínas do Nucleocapsídeo de Coronavírus/genética , Microscopia Crioeletrônica , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Conformação ProteicaRESUMO
An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption 1,2 . There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.