ACE2基因敲除HEK293细胞

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.

BACKGROUND:The COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. METHODS:To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings. RESULTS:While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. CONCLUSIONS:Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.

Angiotensin-converting enzyme 2 (ACE2) is the primary entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but ACE2-independent entry has been observed in vitro for strains with the spike-E484D substitution. Here, we conduct a whole-genome CRISPR-Cas9 knockout screen using SARS-CoV-2 mouse adapted 1 (SARS-CoV-2), which carries spike-E484D, to identify the ACE2-independent entry mechanisms. SARS-CoV-2 infection in HEK293T cells relies on heparan sulfate and endocytic pathways, with TMEM106B, a transmembrane lysosomal protein, the most significant contributor. While SARS-CoV-2 productively infects human brain organoids and K18-hACE2 mouse brains, it does not infect C57BL/6J or Ifnar mouse brains. This suggests that ACE2-independent entry via TMEM106B, which is predominantly expressed in the brain, does not overtly increase the risk of SARS-CoV-2 neuroinvasiveness in mice with endogenous Ace2 expression. Importantly, SARS-CoV-2 does not replicate in the Ace2 mouse respiratory tract. Overall, this suggests that robust ACE2-independent infection by SARS-CoV-2 is likely an in vitro phenomenon with no apparent implications for infection in vivo.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.

Type I interferons exert their antiviral effects against SARS-CoV-2 by inducing the expression of interferon-stimulated genes (ISGs), including but not limited to LY6E, CH25H, IFITM2/3, and IFIH1. However, the antiviral effect and underlying mechanisms of action of most ISGs in SARS-CoV-2 infection are not yet fully understood. By screening 109 ISG-knockout cell lines, we identify that phospholipid scramblase 1 (PLSCR1), an interferon-inducible protein, acts as a crucial restriction factor against SARS-CoV-2 infection. Cells lacking PLSCR1 are highly susceptible to SARS-CoV-2 infection. Conversely, overexpression of PLSCR1 inhibits SARS-CoV-2 infection. Depletion of PLSCR1 enhances cellular entry of both pseudotyped and authentic SARS-CoV-2. Mechanistically, PLSCR1 inhibits SARS-CoV-2 entry by specifically downregulating plasma membrane expression of ACE2, the virus's receptor, without affecting the overall levels of ACE2 within the cell. As such, we unraveled previously unappreciated mechanisms by which PLSCR1 exerts its restrictive effect on SARS-CoV-2. These data provide new insights into the interplay between host innate antiviral immunity and SARS-CoV-2 and shed light on novel antiviral therapeutics. IMPORTANCE:Phospholipid scramblase 1 (PLSCR1) has been identified as a critical host restriction factor against SARS-CoV-2 infection. In this study, we demonstrated that PLSCR1 inhibited SARS-CoV-2 entry by downregulating the plasma membrane expression of ACE2, the primary receptor for viral entry. Our findings elucidate a novel host-pathogen interaction that not only deepens our understanding of the innate immune response to SARS-CoV-2 but offers potential strategies for therapeutic interventions against COVID-19.

SARS-CoV-2 depends on host cell components for infection and replication. Identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection and replication. If druggable, host factor dependencies may present an attractive strategy for anti-viral therapy. In this study, we performed genome wide CRISPR knockout screens in Vero E6 cells and four human cell lines including Calu-3, UM-UC-4, HEK-293 and HuH-7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only , the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while other host genes identified were largely cell line specific, including known factors and . Several of the discovered host-dependency factors converged on pathways involved in cell signalling, immune-related pathways, and chromatin modification. Notably, the chromatin modifier gene in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed.

The global COVID-19 pandemic continues with continued cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus-host dependencies, and characterizing viral variants. Using reverse genetics, we rescued SARS-CoV-2 Wuhan1 (D614G variant) wild type (WTFL) and reporter virus (NLucFL) using molecular BAC clones. The replication kinetics, plaque morphology, and titers were comparable between viruses rescued from molecular clones and a clinical isolate (VIDO-01 strain). Furthermore, the reporter SARS-CoV-2 NLucFL virus exhibited robust luciferase values over the time course of infection and was used to develop a rapid antiviral assay using remdesivir as proof-of-principle. In addition, as a tool to study lung-relevant virus-host interactions, we established novel human lung cell lines that support SARS-CoV-2 infection with high virus-induced cytopathology. Six lung cell lines (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, and HCC827) and HEK293T cells were transduced to stably express ACE2 and tested for their ability to support virus infection. A549ACE2 B1 and HEK293TACE2 A2 cell lines exhibited more than 70% virus-induced cell death, and a novel lung cell line, NCI-H23ACE2 A3, showed about ~99% cell death post-infection. These cell lines are ideal for assays relying on live-dead selection, such as CRISPR knockout and activation screens.