Two mutation (K1269A and H1271A) to eliminate a prospective ER retention domain name) based on data from SARS-CoV1 (McBride et?al., 2007) were introduced by PCR. data of the CRISPR KO screens reported in this paper is usually EMBL-EBI ArrayExpress: E-MTAB-9638. Abstract The are a family of viruses that cause disease in humans ranging from moderate respiratory contamination 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 Diosgenin glucoside 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 contamination by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 contamination. 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. family includes seven known human pathogens for which there are no approved vaccines and only limited therapeutic options. The seasonally circulating human coronaviruses (HCoV) OC43, HKU1, 229E, and NL63 cause moderate, common cold-like respiratory infections in humans (van der Hoek, 2007). However, three highly pathogenic coronaviruses emerged in the last two decades, highlighting the pandemic potential of this viral family (Drosten et?al., 2003; Wu et?al., 2020; Zaki et?al., 2012). Contamination with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV) can lead to acute respiratory distress syndrome and death, with fatality rates between 10%C40% (Petersen et?al., 2020). SARS-CoV-2, though less deadly, is usually far more transmissible than SARS-CoV-1 and MERS-CoV and has been responsible for over 50 million cases and 1.2 million deaths globally as of November 2020 (Dong et?al., 2020; Petersen et?al., 2020). Because of the severity of their impact on global health, it is critical to understand how SARS-CoV-2 and other coronaviruses hijack the host cell machinery during contamination and apply this knowledge to develop new therapeutic strategies. Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with a genome length of approximately 30 kb. Upon?receptor binding and membrane fusion, the viral RNA is released into the cytoplasm, where it is translated to produce viral?proteins. Subsequently, the viral replication/transcription complexes form on double-membrane vesicles and generate genome copies. These are then packaged into new virions via a budding process, through which they acquire the viral envelope, and the resulting virions are released from infected cells (Fung and Liu, 2019). During these actions, specific cellular proteins are hijacked and play crucial roles in the viral life cycle. For example, the angiotensin-converting enzyme 2 (ACE2) is usually exploited as the viral entry receptor for NL63, SARS-CoV-1, and SARS-CoV-2 (Hofmann et?al., 2005; Letko et?al., 2020; Li et?al., 2003). Additionally, cellular proteases, such as TMPRSS2, cathepsin L, and furin, are important for the cleavage of the viral spike (S) protein of several coronaviruses Ptgfrn thereby mediating efficient membrane fusion with host cells (Bertram et?al., 2013; Hoffmann et?al., 2020b; 2020c; Shirato et?al., 2013; Simmons et?al., 2005). Systematic studies have illuminated virus-host interactions during the later actions of the viral life cycle. For example, proteomics approaches revealed comprehensive interactomes between individual coronavirus proteins and cellular proteins (Gordon et?al., 2020a; 2020b; Stukalov et?al., 2020). Additionally, biotin labeling identified candidate host factors based on their proximity to coronavirus replicase complexes (Vkovski et?al., 2019). While these studies uncovered physical relationships between viral and cellular proteins, they do not provide immediate information about the importance of these host components for viral replication. An orthogonal strategy is usually to screen for mutations that render host cells resistant to viral contamination using CRISPR-based mutagenesis. These screens identify host factors that are functionally required for viral contamination and could be targets for host-directed therapies (Puschnik et?al., 2017). In this study, we have performed a genome-wide CRISPR knockout (KO) screen using SARS-CoV-2 (USA/WA-1 isolate) in human cells. Importantly, we expanded our functional genomics approach to distantly related members in order to probe for commonalities and differences across the family. This strategy can reveal potential pan-coronavirus host factors and thus illuminate targets for antiviral therapy to combat the current and potential future outbreaks. We conducted comparative CRISPR screens for SARS-CoV-2 and two seasonally circulating common cold coronaviruses, OC43 and 229E. Our results corroborate previously implicated host pathways, uncover new aspects of virus-host conversation, and identify targets for host-directed antiviral treatment. Results CRISPR KO Screens Identify Common and Virus-Specific Candidate Host Factors for Coronavirus Contamination.We then tested whether the hits shared between OC43 and 229E affect SARS-CoV-2. 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 contamination by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 contamination. 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. family includes seven known human pathogens for which there are no approved vaccines and only limited therapeutic options. The seasonally circulating human coronaviruses (HCoV) OC43, HKU1, 229E, and NL63 cause moderate, common cold-like respiratory infections in humans (van der Hoek, 2007). However, three highly pathogenic coronaviruses emerged in the last two decades, highlighting the pandemic potential of this viral family (Drosten et?al., 2003; Wu et?al., 2020; Zaki et?al., Diosgenin glucoside 2012). Contamination with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV) can lead to acute respiratory distress syndrome and death, with fatality rates between 10%C40% (Petersen et?al., 2020). SARS-CoV-2, though less deadly, is usually far more transmissible than SARS-CoV-1 and MERS-CoV and has been responsible for over 50 million cases and 1.2 million deaths globally as of November 2020 (Dong et?al., 2020; Petersen et?al., 2020). Because of the severity of their impact on global health, it is critical to understand how SARS-CoV-2 and other coronaviruses hijack Diosgenin glucoside the host cell machinery during infection and apply this knowledge to develop new therapeutic strategies. Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with a genome length of approximately 30 kb. Upon?receptor binding and membrane fusion, the viral RNA is released into the cytoplasm, where it is translated to produce viral?proteins. Subsequently, the viral replication/transcription complexes form on double-membrane vesicles and generate genome copies. These are then packaged into new virions via a budding process, through which they acquire the viral envelope, and the resulting virions are released from infected cells (Fung and Liu, 2019). During these steps, specific cellular proteins are hijacked and play crucial roles in the viral life cycle. For example, the angiotensin-converting enzyme 2 (ACE2) is exploited as the viral entry receptor for NL63, SARS-CoV-1, and SARS-CoV-2 (Hofmann et?al., 2005; Letko et?al., 2020; Li et?al., 2003). Additionally, cellular proteases, such as TMPRSS2, cathepsin L, and furin, are important for the cleavage of the viral spike (S) protein of several coronaviruses thereby mediating efficient membrane fusion with host cells (Bertram et?al., 2013; Hoffmann et?al., 2020b; 2020c; Shirato et?al., 2013; Simmons et?al., 2005). Systematic studies have illuminated virus-host interactions during the later steps of the viral life cycle. For example, proteomics approaches revealed comprehensive interactomes between individual coronavirus proteins and cellular proteins (Gordon et?al., 2020a; 2020b; Stukalov et?al., 2020). Additionally, biotin labeling identified candidate host factors based on their proximity to coronavirus replicase complexes (Vkovski et?al., 2019). While these studies uncovered physical relationships between viral and cellular proteins, they do not provide immediate information about the importance of these host components for viral replication. An orthogonal strategy is to screen for mutations that render host cells resistant to viral infection using CRISPR-based mutagenesis. These screens identify host factors that are functionally required for viral infection and could be targets for host-directed therapies (Puschnik et?al., 2017). In this study, we have performed a genome-wide CRISPR knockout (KO) screen using SARS-CoV-2 (USA/WA-1 isolate) in human cells. Importantly, we expanded our functional genomics approach to distantly related members in order to probe for commonalities and differences across the family. This strategy can reveal potential pan-coronavirus host factors and thus illuminate targets for antiviral therapy to combat the current and potential future outbreaks. We conducted comparative CRISPR screens for SARS-CoV-2 and two seasonally circulating common cold coronaviruses, OC43 and 229E. Our results corroborate previously implicated host pathways, uncover new aspects of virus-host interaction, and identify.