key: cord-0690352-wzx95wrb authors: Grodzki, Marco; Bluhm, Andrew P.; Schäfer, Moritz; Tagmount, Abderrahmane; Russo, Max; Sobh, Amin; Rafiee, Roya; Vulpe, Chris D.; Karst, Stephanie M.; Norris, Michael H. title: Genome-scale CRISPR Screens Identify Host Factors that Promote Human Coronavirus Infection date: 2021-06-04 journal: bioRxiv DOI: 10.1101/2021.06.04.447090 sha: bd56470d1f9548d55b5f08fe422a84d3eacf1ba2 doc_id: 690352 cord_uid: wzx95wrb The COVID-19 pandemic has resulted in 153 million infections and 3.2 million deaths as of May 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as potentially antigenically distinct 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. 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. 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. 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. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. 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. One Sentence Summary Genome-wide CRISPR screens identified host factors that promote human coronavirus infection, revealing novel antiviral drug targets. One Sentence Summary: Genome-wide CRISPR screens identified host factors that promote 25 human coronavirus infection, revealing novel antiviral drug targets. 26 27 Abstract: The COVID-19 pandemic has resulted in 153 million infections and 3.2 million deaths 28 as of May 2021. While effective vaccines are being administered globally, there is still a great 29 need for antiviral therapies as potentially antigenically distinct SARS-CoV-2 variants continue to 30 emerge across the globe. Viruses require host factors at every step in their life cycle, representing 31 a rich pool of candidate targets for antiviral drug design. To identify host factors that promote 32 SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, 33 we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T 34 ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human 35 coronavirus OC43. While we identified multiple genes and functional pathways that have been 36 previously reported to promote human coronavirus replication, we also identified a substantial The COVID-19 pandemic is arguably the most consequential infectious disease outbreak in 46 modern times. The causative agent of COVID-19, SARS-CoV-2, spread quickly across the planet 47 resulting in 153 million infections and 3.2 million deaths at the time of this writing. Multiple 48 COVID-19 vaccines recently demonstrated high efficacy, received FDA approval, and are being 49 administered to people across the globe. While the importance of this scientific achievement 50 cannot be understated, there is still a great need for novel antiviral therapies for use in vulnerable 51 immunocompromised individuals, in regions where vaccine access is limited, and in the event that 52 antigenically distinct SARS-CoV-2 variants emerge. Moreover, considering that SARS-CoV-2 is 53 the third novel human coronavirus (HCoV) to emerge and cause serious disease in the human 54 population in the past two decades following SARS-CoV and MERS-CoV, potent and broad-55 spectrum antivirals will leave us better prepared to deal with future pandemics. Broad-spectrum 56 antivirals could also reduce morbidity associated with common cold-causing HCoVs including 57 OC43, NL63, 229E, and HKU1. 58 Antivirals segregate into two basic categories, virus-targeting and host-targeting, both of which 59 require an understanding of the molecular mechanisms used by viruses to replicate in host cells. 60 Coronaviruses replicate via a well-established series of molecular events (1, 2) . Host factors are 61 required at every step in this life cycle and represent candidate druggable targets (i.e. host-targeting 62 antivirals) with the potential for broad-spectrum activity against multiple viruses within a given interactions. 76 In this study, we report a global analysis of host-HCoV interactions gleaned from genome-77 wide screens performed for two HCoVs and in two different cell lines. We also performed a 78 comprehensive comparative analysis of all published genome-wide SARS-CoV-2 screens to date. 79 Multiple genes and functional pathways identified in our screens were previously reported to 80 promote SARS-CoV-2 replication, validating the rigor of our approach and providing further 81 support for the role of specific host factors. Yet we also identified a substantial number of novel 82 genes and pathways not previously reported to promote HCoV replication. We validated the 83 importance of a subset of genes identified in these screens in HCoV replication. Notably, several 84 of the novel host factors identified in our study provide unique insight into SARS-CoV-2 85 replication processes that could be targeted with antiviral drugs. Host factors involved in cell cycle 86 regulation were enriched in our screens and we show that compounds (abemaciclib, AZ1 protease 87 inhibitor, harmine, nintedanib, and UC2288) targeting these host factors inhibit in vitro HCoV 88 replication. We also identified multiple host factors involved in endocytosis and TBK1 that plays 89 a key role in innate immune responses. Inhibitors of these processes/factors (promethazine and 90 amlexanox, respectively) also displayed antiviral activity. The website was designed to facilitate integration of upcoming screens and we hope for 114 contributions to drive this as a community project. 115 We identified multiple candidate host factors previously demonstrated to play a functional 116 role in SARS-CoV-2 and OC43 infections. For example, ACE2 was identified in the SARS-CoV-117 2 screen (11). Furthermore, TMEM41B was a top-scoring gene in the OC43 screen, supporting 118 recent work by Schneider et al. demonstrating that TMEM41B is a pan-HCoV host factor (7). We Study design 345 The objectives of this study were to identify host factors that promote HCoV replication and to 346 determine whether these host factors can be targeted with commercially available drugs to block 347 viral infection in vitro (Fig. S1 ). To achieve this goal, we performed genome-wide CRISPR 348 knockout screens in Vero E6 cells using a newly generated Vervet sgRNA library (Fig. 1A) and TMEM41B CRACR2B ITGB8 APT1 C18orf8 CCZ1 CCZ1B RAB7A WDR81 WDR91 ASNA1 GET4 WRB EXT2 C1GALT1 CASD1 INPP5K TMED2 TMED10 IFITM1 IFITM2 IFITM3 TRAF3IP2 SIGIRR OR10J3 OR52B2 PTAR1 VMP1 VPS16 YBX3 TXN ADNP ARID1A SMARCA4 SMARCB1 SMARCC1 GABPB1 PHRF1 KMT2D ANO9 SEC61B SLC35A1 SLC35B2 SLC35D1 SCARA5 SLC39A9 SLC4A11 CCDC93 DOCK5 KRTAP5-7 MDH1 NXPE3 PANO1 SLC35B1 SUCO TAPT1 TM9SF3 5 10 15 Calcium Signaling C18orf8 CCDC28A CCM2 CCZ1 CDH2 CLTC CTBP2 DAZ3 DECR1 DISP2 DNAH2 EDC4 EPN1 ERGIC3 ERICH6B F8A1 FBXW10 GNPTAB GNPTG GUCY2D IGSF11 INTS12 INTS6 ITFG2 KBTBD6 MESDC1 MPLKIP MTMR9 MYH13 NAGPA PDIA6 PEX10 PKHD1L1 PTCH1 RFX7 RFXAP RNF7 SARNP SEC63 SLC12A9 SLC39A1 SMCHD1 STRADA TBC1D7 THAP7 TMEM11 TMEM39A TP53 URAD WDR35 XRN1 ACE2 ALG5 ARVCF ATP6V1A ATP6V1G1 B3GAT3 CCZ1B CNOT4 CTSL DNM2 EPT1 EXOC2 EXT1 EXTL3 GDI2 LUC7L2 MBTPS2 PIK3C3 RAB7A RNH1 SCAF4 SCAP SLC30A1 SLC33A1 SNX27 TMEM106B TMEM251 TMEM41B VAC14 VPS35 WDR81 WDR91 1 2 3 4 The 517 molecular virology of coronaviruses Altan-Bonnet, β-Coronaviruses Use Lysosomes for 521 HSP90: a promising broad-523 spectrum antiviral drug target The future of antivirals: broad-spectrum inhibitors A genome-wide 528 CRISPR screen identifies host factors that regulate SARS-CoV-2 entry Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses Genome-Scale Identification of SARS-CoV-2 and Pan-538 coronavirus Host Factor Networks Genome-wide CRISPR Screens Reveal Host 544 Factors Critical for SARS-CoV-2 Infection Identification of Required Host Factors for SARS-548 CoV-2 Infection in Human Cells Identification of TMEM106B as proviral host factor for 551 SARS-CoV-2 CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven 555 Interferon induction 557 of IFITM proteins promotes infection by human coronavirus OC43 Opposing activities of IFITM proteins 561 in SARS-CoV-2 infection The polybasic cleavage site in the SARS-CoV-2 spike modulates 564 viral sensitivity to Type I interferon and IFITM2 CRISPR screens with MAGeCK-VISPR Optimized sgRNA 570 design to maximize activity and minimize off-target effects of CRISPR-Cas9 Characterization of spike glycoprotein of 574 SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV A Crisp(r) New Perspective on SARS-CoV-2 Biology 579 RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway Targeting TANK-binding kinase 1 (TBK1) in cancer Reveals the Requirement of Host Cell Sulfation for Schmallenberg Virus Infection Role of heparan 590 sulfate in the Zika virus entry, replication, and cell death A genome-wide CRISPR 594 screen identifies a restricted set of HIV host dependency factors Chronic phospholamban-sarcoplasmic reticulum calcium 598 ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy Role of dimerized C16orf74 in aggressive pancreatic 602 cancer: a novel therapeutic target Overexpression of C16orf74 is involved in aggressive pancreatic cancers Rotavirus Induces Formation of Remodeled Stress Granules and P 607 Bodies and Their Sequestration in Viroplasms To Promote Progeny Virus Production Stress Granules, and Viral Life Cycles Human coronaviruses disassemble processing bodies, bioRxiv Identifying SARS-CoV-2 Entry Inhibitors through Drug Repurposing Screens of 616 SARS-S and MERS-S Pseudotyped Particles Identification of antiviral antihistamines for COVID-19 repurposing Added value of an oropharyngeal swab in detection of viruses in children 623 hospitalized with lower respiratory tract infection Molecular 625 Identification and Functional Characterization of the Human Colonic Thiamine Pyrophosphate 626 Genome-Scale CRISPR-Cas9 Knockout Screening in 629 Human Cells MAGeCK enables robust identification of essential genes from genome-scale 632 CRISPR/Cas9 knockout screens The genome of the vervet (Chlorocebus aethiops 642 sabaeus) FlashFry: a fast and flexible tool for large-scale CRISPR target 644 design PAVOOC: designing CRISPR sgRNAs 646 using 3D protein structures and functional domain annotations MAGeCK enables robust identification of essential genes from genome-scale 650 CRISPR/Cas9 knockout screens Unbiased and Tailored CRISPR/Cas gRNA Libraries 652 by Synthesizing Covalently-closed-circular (3Cs) DNA Genome-scale CRISPR-Cas9 knockout and transcriptional activation 655 screening The COVID-19 Drug and Gene Set Library Acknowledgments: We thank Dr This work was supported by the UF Clinical and 660 Funding acquisition: MHN, SMK, CDV 666 Project administration: MHN, SMK, CDV 667 Supervision: MHN, SMK, CDV 668 Writing -original draft: MG, APB 669 Writing -review & editing: MHN, SMK, CDV 670 Competing interests: A patent entitled Methods of Treatment for SARS-CoV-2 Infections 671 Data and materials availability: All data are available in the main text or the supplementary Vero E6 694 cells transduced with a C. sabaeus-specific sgRNA library were infected with SARS-CoV-2 or 695 OC43 at MOI 0.01, resistant cells reinfected at MOI 0.01, and sgRNAs in resistant clones 696 sequenced. MAGeCK analysis of multiple replicates compared to uninfected control library 697 replicates yielded log2fold changes (log2FC) that were plotted on the x-axis OC43 (B) Vero E6 infections. The heat maps display the log2FC for the 20 top-700 scoring genes comparing results for SARS-CoV-2 and OC43 infections Fig 5. Comparison of multiple CRISPR screens identifying host factors promoting SARS-730 CoV-2 infection of human cell lines. A) Data from four recently published CRISPR screens for 731 Using this criterion, there were 74 genes identified in our 733 study, 53 in Daniloski et al., 707 in Schneider et al., 13 in Wang et al., and 1 in Baggen et al. No 734 common genes were identified in all studies, 1 gene was identified in four studies, 6 genes were 735 identified in three studies, and 25 genes were identified in two studies. 53 genes were uniquely 736 identified in our study as significant. B) The heat map displays the log2FC for the 32 genes found 737 in common across two or more of the published studies with FDR<0.25. C) A heat map displaying 738 the log2FC for the 53 genes uniquely identified as Lentivirus-743 packaged shRNA clones directed to CTSL, CCZ1, and EDC4 were transduced into HEK293T-744 hACE2 cells and selected with puromycin. A) Gene knockdown was assessed using western 745 blotting with antibodies directed to CTSL, CCZ1, and EDC4 in cells transduced with a gene-746 specific shRNA or empty vector control (EV) At 2 748 dpi, viral genome copy numbers were determined by RT-qPCR and normalized to GAPDH levels 749 as a housekeeping control. The data are reported as the relative normalized viral genome copy 750 number in shRNA-expressing cells compared to the EV control (n = 3 experiments). Error bars 751 denote standard errors of mean and P values were determined using one-way ANOVA The 757 log2FC across the studies performed in this work for the 12 gene targets that had commercially 758 available inhibitors. The + and -signs to the right of the heat map summarize the ability of these 759 small molecules to prevent SARS-CoV-2 infection of Vero E6 cells. B) Initial screening of drug 760 inhibition of SARS-CoV-2 Drug inhibition of SARS-CoV-2 genome replication was measured by RT-qPCR at 2 dpi of Vero 762 E6 cells at MOI 0.01. One-way ANOVA was used to compare inhibitor-treated toxicity values to 763 virus-alone controls. D-J) The EC50 (ability of inhibitors to reduce SARS IC50 (toxicity of inhibitors alone) curves were obtained by cytotoxicity assays in Vero E6 cells Error bars 766 indicate standard deviation for all panels (n = 3). 767 no Virus SARS-CoV-2 Summary of genes found in this and other studies and their potential roles in the 769 The host factors identified in CRISPR screens are presented adjacent to 770 the putative stage of viral replication where they function. The genes are color-coded based on 771 their identification in our and other published studies, as The virus replicates through a series of well-773 defined molecular steps. 1-2) After virion binding to ACE2, SARS-CoV-2 can fuse at the plasma 774 membrane or following endocytosis. Heparan sulfate proteoglycans enhance viral attachment to 775 cells so host factors The IFITM 777 proteins are proposed to promote fusion at the cell surface but inhibit fusion in endosomes Certain host factors like RNH1 and DAZ3 may serve to protect the viral genome 784 from degradation by host enzymes. 4) The nsps form the viral replicase which assembles on 785 organellar membranes to form the replication and transcription complexes (RTCs) where progeny 786 genomes and structural/accessory protein transcripts are produced, respectively. P-body 787 components EDC4 and XRN1, identified in this study, may play a role in the maintaining viral 788 RNA stability or assembly of the RTC. 5) Structural and accessory proteins are translated, and 789 structural proteins insert into the ER membrane 7) Progeny virions form as they traverse through the Golgi and structural 792 proteins are glycosylated. 8) Virions exit the cell through either typical exocytosis VPS35) or nonclassical lysosomal egress Numerous host factors with less obvious direct 795 roles in promoting steps in the viral life cycle have also been identified in CRISPR screens TP53) were identified in our screens in AGM and human cells Furthermore, multiple nuclear-localized host factors including diverse transcriptional regulators 799 and two components of the integrator complex (INTS6, INTS12) were identified. Overall, the large 800 number of diverse host factors that promote SARS-CoV-2 replication illustrates the large-scale 801 exploitation of cellular processes required for successful viral propagation BioRender template titled Life Cycle of Coronavirus generated by the Britt Glaunsinger 803 laboratory