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Genome-Wide CRISPR Screening Identifies TMEM106B As a Proviral Host Factor for SARS-Cov-2

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Genome-Wide CRISPR Screening Identifies TMEM106B As a Proviral Host Factor for SARS-Cov-2 ARTICLES https://doi.org/10.1038/s41588-021-00805-2 Genome-wide CRISPR screening identifies TMEM106B as a proviral host factor for SARS-CoV-2 Jim Baggen 1 ✉ , Leentje Persoons 1,8, Els Vanstreels 1,8, Sander Jansen 1,8, Dominique Van Looveren 1,2, Bram Boeckx3,4, Vincent Geudens 5, Julie De Man1, Dirk Jochmans 1, Joost Wauters6, Els Wauters 5, Bart M. Vanaudenaerde5, Diether Lambrechts3,4, Johan Neyts1, Kai Dallmeier 1, Hendrik Jan Thibaut 1,2, Maarten Jacquemyn 1, Piet Maes7 and Dirk Daelemans 1 ✉ The ongoing COVID-19 pandemic has caused a global economic and health crisis. To identify host factors essential for corona- virus infection, we performed genome-wide functional genetic screens with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E. These screens uncovered virus-specific as well as shared host factors, including TMEM41B and PI3K type 3. We discovered that SARS-CoV-2 requires the lysosomal protein TMEM106B to infect human cell lines and primary lung cells. TMEM106B overexpression enhanced SARS-CoV-2 infection as well as pseudovirus infection, sug- gesting a role in viral entry. Furthermore, single-cell RNA-sequencing of airway cells from patients with COVID-19 demonstrated that TMEM106B expression correlates with SARS-CoV-2 infection. The present study uncovered a collection of coronavirus host factors that may be exploited to develop drugs against SARS-CoV-2 infection or future zoonotic coronavirus outbreaks. he COVID-19 pandemic, caused by SARS-CoV-2, has to address the current high medical need, but also to quickly con- resulted in a worldwide health crisis1 and few effective drugs tain zoonotic events in the future. Common host factors essential are available to treat patients with COVID-19. Although for replication of multiple coronaviruses represent attractive targets T 2 remdesivir initially seemed promising for severe cases , the World for broad-spectrum antiviral drugs. Health Organization’s Solidarity trial showed that it has no definite To develop such drugs, it is crucial to understand which host impact on mortality3. Dexamethasone can reduce mortality by a factors coronaviruses require to infect a cell, because each step of third among critically ill patients with COVID-19, by suppressing the coronavirus replication cycle (receptor binding, endocytosis, the hyperactive immune response4. However, as treatment benefits fusion, viral protein translation, genome replication, virion assem- severe cases only to a limited extent, efficient and safe therapeutics bly and release) may serve as a target for intervention. Although the are urgently required while awaiting the worldwide implementation entry step of coronaviruses has been relatively well characterized, of vaccines. the host–virus interplay in later steps of the viral life cycle remains Coronaviruses cause respiratory and intestinal infections in a largely elusive. For SARS-CoV-2, previous studies have shown that broad range of mammals and birds. Seven human coronaviruses the protein angiotensin-converting enzyme 2 (ACE2) can serve as (HCoVs) are known, which probably all emerged as zoonoses from a receptor in Vero E6 cells9 or in human cells overexpressing ACE2 bats, mice or domestic animals5. The four so-called ‘common cold (refs. 10–12). In addition, it was shown that the SARS-CoV-2 spike HCoVs’—229E, NL63, OC43 and HKU1—cause mild upper respi- (S) can be primed for fusion by cellular proteases such as furin, ratory tract illnesses6. In contrast, SARS-CoV, Middle East respira- transmembrane serine protease 2 (TMPRSS2) or cathepsin B or L, tory syndrome coronavirus (MERS-CoV) and the recently emerged depending on the target cell type10,13. SARS-CoV-2 are highly pathogenic and cause severe, potentially In the present study, we performed a series of genome-wide lethal respiratory infections. As numerous coronaviruses reside CRISPR (clustered regularly interspaced short palindromic in animal reservoirs and interspecies transmission frequently repeats)-based genetic screens to identify host factors required occurs5,7,8, there is a constant risk of new pathogenic coronaviruses for SARS-CoV-2 and HCoV-229E infection. We identified phos- spreading into the human population, as exemplified by the recent phoinositide 3-kinase (PI3K) type 3 as a common host factor for SARS-CoV-2 pandemic. Nevertheless, our options to prevent or SARS-CoV-2, HCoV-229E and HCoV-OC43, and show that small treat coronavirus infections remain limited. Hence, the develop- molecules targeting this protein might serve as broadly applicable ment of broad-spectrum anti-coronavirus drugs could help not only anti-coronavirus inhibitors. Furthermore, we discovered that the 1KU Leuven Department of Microbiology, Immunology and Transplantation, Laboratory of Virology and Chemotherapy, Rega Institute, Leuven, Belgium. 2KU Leuven Department of Microbiology, Immunology and Transplantation, Laboratory of Virology and Chemotherapy, Translational Platform Virology and Chemotherapy, Rega Institute, Leuven, Belgium. 3KU Leuven Department of Human Genetics, Laboratory for Translational Genetics, Leuven, Belgium. 4VIB Center for Cancer Biology, VIB, Leuven, Belgium. 5KU Leuven Department of Chronic Diseases and Metabolism, Laboratory of Respiratory Diseases and Thoracic Surgery (BREATHE), Leuven, Belgium. 6KU Leuven Department of Microbiology, Immunology and Transplantation, Laboratory of Clinical Infectious and Inflammatory Disorders, Leuven, Belgium. 7KU Leuven Department of Microbiology, Immunology and Transplantation, Laboratory of Clinical and Epidemiological Virology, Rega Institute, Leuven, Belgium. 8These authors contributed equally: Leentje Persoons, Els Vanstreels, Sander Jansen. ✉e-mail: [email protected]; [email protected] NatURE GENETICS | VOL 53 | APRIL 2021 | 435–444 | www.nature.com/naturegenetics 435 ARTICLES NATURE GENETICS a Huh7 cells Knockout cell pool Resistant knockout cells sgRNA sequencing Lentiviral Brunello SARS-CoV-2 or sgRNA library HCoV-229E b d 100.0 HCoV-229E 5.5 SARS-CoV-2 low stringency ANPEP Overlapping between HCoV-229E TMEM41B and SARS-CoV-2 10.0 5.0 AHCYL1 Membrane/vesicle trafficking PIK3C3 Lipid homeostasis EDRF1 NUFIP2 STAT2 TAS1R1 1.0 4.5 Known viral receptor (synthesis) gene KRT19 0.10 4.0 GLUD1 Enrichment (fold-change) DTD1 PTDSS1 OSBPL9 c17orf53 CCZ1B –log(P) = 1 SALL2 0.01 Gene 3.5 TMEM30A –log(P) = 0.1 EXT1 CEP350 c MAPKAPK3 RPL19 100.0 SARS-CoV-2 high stringency PTPN23 c7orf77 ITGB6 RSG1 3.0 PIK3C3 TMEM41B TMEM106B Enrichment (fold-change) 10.0 2.5 ACE2 TMEM106B 1.0 ACE2 2.0 1.5 0.10 Enrichment (fold-change) 1.0 0.5 –log(P) = 0.5 Gene Gene 0.01 –log(P) = 0.1 0 –log(P) = 3 –log(P) = 2 –log(P) = 1 Fig. 1 | Genome-wide knockout screens in human cells identify host factors for SARS-CoV-2 and HCoV-229E infection. a, Overview of experimental steps performed during a genome-wide screen for coronavirus host factors. b–d, Genome-wide knockout screens performed in Huh7 cells, with strong selection (high stringency) using HCoV-229E (b) and SARS-CoV-2 (c) or with mild SARS-CoV-2 selection (low stringency) (d). Each circle represents a gene, with size corresponding to significance (−log(P value)) of enrichment. The enrichment (y axis) represents the average change in abundance of the four sgRNAs for each gene, when comparing sgRNA abundances between the virus-selected population and the sgRNA library before selection (b and c) or between the virus-selected population and an uninfected control population (d). Genes are distributed on the x axis in alphabetical order. The sgRNA-level P values were calculated using a one-sided Student’s t-test and aggregated using Fisher’s method to obtain gene-level P values44. Data result from two independent screen replicates. lysosomal protein TMEM106B serves as an essential specific host we also performed a lower-stringency screen (Fig. 1d) to identify factor for SARS-CoV-2 infection in multiple liver- and lung-derived genes having a more subtle effect on virus infection. human cell lines. As expected, the HCoV-229E screen identified ANPEP (Fig. 1b), encoding the established HCoV-229E receptor aminopeptidase N Results (AP-N)21. Our SARS-CoV-2 screens did not identify ACE2, in con- Genome-wide knockout screens for HCoV-229E and SARS- trast to screens performed in Vero E6 cells9 or ACE2-overexpressing CoV-2. Genome-wide knockout screens have been widely used to human cells12,16,18. This is probably due to the low ACE2 expression identify host factors for various viruses14,15 and were recently per- in Huh7 cells compared with Vero E6 or ACE2-overexpressing cells22 formed with coronaviruses, including SARS-CoV-2 (refs. 9,12,16–19). (Extended Data Fig. 1). The high-stringency SARS-CoV-2 screen Most of these screens were performed in human cells that were engi- (Fig. 1c) identified one significantly enriched gene, TMEM106B, neered to overexpress ACE2 (refs. 12,16,18,19). In the present study, we encoding the poorly characterized protein TMEM106B, which performed a CRISPR-based genome-wide knockout screen in the is involved in lysosome function and implicated in neurodegen- human Huh7 cell line, without introducing an exogenous receptor, erative disorders23. A larger number of genes were enriched in because our SARS-CoV-2 strain induced a clear cytopathic effect the low-stringency screen (Fig. 1d). However, a low-stringency (CPE) in these cells. We performed screens with both SARS-CoV-2 screen may have an increased background due to the presence of and the less pathogenic HCoV-229E. This allowed for the identifi- a subpopulation of cells that are not infected and may be selected cation of both (1) broad-spectrum coronavirus host factors and (2) for resistance to antiviral stress signals rather than resistance to specific host factors for SARS-CoV-2 and HCoV-229E. Huh7 cells virus. Two genes were present among the top-ranked genes in transduced with the Brunello genome-wide library20 were selected both the HCoV-229E and the SARS-CoV-2 low-stringency screen for survival during infection with either coronavirus.
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