RESUMEN
The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.
Asunto(s)
COVID-19/genética , Infecciones por Coronavirus/genética , Coronavirus/fisiología , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , SARS-CoV-2/fisiología , Células A549 , Animales , Vías Biosintéticas/efectos de los fármacos , COVID-19/virología , Línea Celular , Chlorocebus aethiops , Colesterol/biosíntesis , Colesterol/metabolismo , Análisis por Conglomerados , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Resfriado Común/genética , Resfriado Común/virología , Coronavirus/clasificación , Infecciones por Coronavirus/virología , Técnicas de Inactivación de Genes , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Ratones , Fosfatidilinositoles/biosíntesis , Células Vero , Internalización del Virus/efectos de los fármacos , Replicación ViralRESUMEN
To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.
Asunto(s)
COVID-19/genética , COVID-19/virología , Interacciones Huésped-Patógeno , SARS-CoV-2/fisiología , Células A549 , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Vías Biosintéticas , COVID-19/metabolismo , Colesterol/biosíntesis , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endosomas/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes/métodos , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Interferencia de ARN , SARS-CoV-2/crecimiento & desarrollo , Análisis de la Célula Individual , Carga Viral/efectos de los fármacos , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Sterol regulatory element binding protein-2 (SREBP-2) is activated by cytokines or pathogen, such as virus or bacteria, but its association with diminished cholesterol levels in COVID-19 patients is unknown. Here, we evaluated SREBP-2 activation in peripheral blood mononuclear cells of COVID-19 patients and verified the function of SREBP-2 in COVID-19. Intriguingly, we report the first observation of SREBP-2 C-terminal fragment in COVID-19 patients' blood and propose SREBP-2 C-terminal fragment as an indicator for determining severity. We confirmed that SREBP-2-induced cholesterol biosynthesis was suppressed by Sestrin-1 and PCSK9 expression, while the SREBP-2-induced inflammatory responses was upregulated in COVID-19 ICU patients. Using an infectious disease mouse model, inhibitors of SREBP-2 and NF-κB suppressed cytokine storms caused by viral infection and prevented pulmonary damages. These results collectively suggest that SREBP-2 can serve as an indicator for severity diagnosis and therapeutic target for preventing cytokine storm and lung damage in severe COVID-19 patients.