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Authors:Wei et al 

Journal/ Pre-Print:bioRxiv 

Tags: Bioinformatics, Cell Biology, Drug discovery/Drug repurpose, Molecular biology, Virology 

Research Highlights 

  1. CRISPR screen in Vero-E6 cells found novel pro-viral genes and pathways (SWI/SNF chromatin remodelling complex, TGF-b signalling pathway) and anti-viral genes (histone H3.3 chaperone complex). 

  1. Small molecule inhibitors of Cathepsin L (Calpain Inhibitor III), SMARCA4 (PFI-3), and SMAD3 (SIS3) protect cells from SARS-CoV-2 infection and induced cell death. 

  1. HMGB1 has been identified as a novel player in SARS-CoV-2 susceptibility. 

Summary 

The authors performed the first genome-wide SARS-CoV-2 CRISPR screen in Vero-E6 cells to discover pro- or anti-viral host genes and pathways. They found that the SWI/SNF chromatin remodelling complex and the TGF-b signalling pathway are pro-viral whereas the histone H3.3 chaperone complex is anti-viral. The secreted alarmin HMGB1 was validated for its role in viral replication. The authors also tested four small molecule inhibitors against SARS-CoV-2 infection and induced cell death and found that three of them, Cathepsin L (Calpain Inhibitor III), SMARCA4 (PFI-3), and SMAD3 (SIS3), offer some protection in Vero-E6 cells. 

Impact for SARS-CoV2/COVID19 research efforts  

Understand the virology and/or cell biology of SARS-CoV2/COVID19 

- new host genes and their pathways have now been revealed, and the genome-wide screen gives an comprehensive picture of how cells respond to the virus, which will have significant impact on understanding the virology and the pathogenesis. 

Develop a vaccine for SARS-CoV2/COVID19 

- Host genes involved in viral replication could be taken into consideration to improve the vaccine production when cell lines are engineered. 

Treat of SARS-CoV2/COVID19 positive individuals 

- A variety of small molecules have been tested effective to protect cells from the virus induced death, which may help treat the disease if they are developed into drugs. 

Study Type  

  • In silico study / bioinformatics study 

  • In vitro study 

Strengths and limitations of the paper 

Novelty: First CRISPR screen with SARS-CoV-2 in Vero-E6 cells where the authors found novel pro-viral genes and pathways (SWI/SNF chromatin remodelling complex, TGF-b signalling pathway) and anti-viral genes (histone H3.3 chaperone complex). The top 50 resistance and sensitization genes found in the CRISPR screen improved the predictability of a single cell of the primary human bronchial epithelial to be infected by SARS-CoV-2. The authors also show in vitro and in one cell line that small molecule inhibitors of Cathepsin L (Calpain Inhibitor III), SMARCA4 (PFI-3), and SMAD3 (SIS3) protect cells from SARS-CoV-2 infection and induced cell death. 

Standing in the field:Known host gene such as ACE2 was also identified to be top hit in the study, but TMPRSS2 was not. Low overlap between the results of the CRISPR screen and a previously published SARS-CoV-2 protein interactome (only three genes overlapping: TIMM10, AP2M1, PIGO). However, the two studies have been performed on two different cell lines and with two different techniques which are not answering exactly the same question (host genes required for SARS-CoV-2 infection and induced cell death for the CRISPR screen and the interactomes of each of SARS-CoV-2 protein for Gordon et al, 2020, Nature). 

The authors did not observe a protective effect against SARS-CoV-2 induced cell death of the PIKFyve kinase inhibitor APY0201 found by Riva et al, 2020, bioRxiv.   

Appropriate statistics: Yes 

Viral model used:SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources #NR-52281); icSARS-CoV-2-mNG 

Translatability:Small molecule inhibitors of Cathepsin L (Calpain Inhibitor III), SMARCA4 (PFI-3), and SMAD3 (SIS3) protect cells from SARS-CoV-2 infection and induced cell death. However, this has been tested only in vitro in one cell line so more work is required. 

Main limitations: 

1) The small molecules inhibitors used in this study were only tested in vitro and in one cell line so their efficiencies in vivo remain to be determined.   

2) The cell line used by the authors, Vero-E6, is a type I interferon-deficient non-human primate cell line, which could therefore limit the discovery of host genes involved in SARS-CoV-2 infection (typically the genes regulated by type I interferon). 

3) Does the lentiviral infection used for library generation have any effect on the responses of cells to SARS-CoV-2? 

4) The total number and the list of genes found to be pro- or anti-viral are not provided. 

5) It remains unclear how the genes found in the CRISPR screen protect or sensitize the cells to SARS-CoV-2 infection and induced cell death. The hypothesis would be that the effect is mediated through regulation of gene expression but no experiments have been performed to test it.