Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

  1. Ruofan Wang
  2. Camille R. Simoneau
  3. Jessie Kulsuptrakul
  4. Mehdi Bouhaddou
  5. Katherine Travisano
  6. Jennifer M. Hayashi
  7. Jared Carlson-Stevermer
  8. Jennifer Oki
  9. Kevin Holden
  10. Nevan J. Krogan
  11. Melanie Ott
  12. Andreas S. Puschnik

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Abstract

The Coronaviridae are a family of viruses that causes disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors that are common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted parallel genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two 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 discovered phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle as well as the potential development of host-directed therapies.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.24.312298: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationContamination: Cell lines were tested negative for mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies were detected by incubating membranes with 1:5000 dilution of HRP-conjugated (Southern Biotech) secondary anti-mouse and anti-rabbit antibodies for 1 h at room temperature.
    anti-mouse
    suggested: None
    anti-rabbit
    suggested: None
    The following primary antibodies and their dilutions were used in this study: GAPDH (SCBT, sc-32233) at 1:1000, ACE2 (R&D Systems, AF933) at 1:1000, TMPRSS2 (Abcam, ab92323) at 1:1000.
    GAPDH
    suggested: (Santa Cruz Biotechnology Cat# sc-32233, RRID:AB_627679)
    ACE2
    suggested: (GenWay Biotech Inc. Cat# 18-661-15169-0.1 mg, RRID:AB_514759)
    TMPRSS2
    suggested: (Abcam Cat# ab92323, RRID:AB_10585592)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: Huh7.5.1 (gift from Frank Chisari), HEK293FT (Thermo Scientific), Vero cells (ATCC) and A549-ACE2 cells (gift from Olivier Schwartz) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Omega Scientific), penicillin/streptomycin (Gibco), non-essential amino acids (Gibco) and L-glutamine (Gibco) at 37C and 5% CO2.
    A549-ACE2
    suggested: None
    Lentivirus was produced in HEK293FT by co-transfection of cDNA containing lentiviral plasmid together with pCMV-dR8.2 dvpr (Addgene, #8455, gift from Bob Weinberg), pCMV-VSV-G (Addgene, #8454, gift from Bob Weinberg) and pAdVAntage (Promega) using FugeneHD (Promega).
    HEK293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    Viral titers were determined by standard plaque assay using either Huh7.5.1 cells (OC43 and 229E) or Vero cells (SARS-CoV-2).
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Genome-wide CRISPR screens: Huh7.5.1-Cas9 cells were generated by lentiviral transduction with lentiCas9-blast (Addgene, #52962, gift from Feng Zhang) and subsequently selected with blasticidin for 7 days.
    Huh7.5.1-Cas9
    suggested: None
    To generate CRISPR KO libraries, a total of 240 million Huh7.5.1-Cas9-blast or Huh7.5.1-Cas9-blast+ACE2-IRES-TMPRSS2-hygro cells were transduced with lentivirus of the human GeCKO v2 library (Addgene, #1000000049, gift from Feng Zhang) at a moi of 0.4 and subsequently selected using puromycin and expanded for 7 days.
    Huh7.5.1-Cas9-blast
    suggested: None
    Huh7.5.1-Cas9-blast+ACE2-IRES-TMPRSS2-hygro
    suggested: None
    Cell viability assay: Huh7.5.1 cells were treated with compounds at the same concentrations and durations as in infection assays.
    Huh7.5.1
    suggested: RRID:CVCL_E049)
    Experimental Models: Organisms/Strains
    SentencesResources
    Cell culture: Huh7.5.1 (gift from Frank Chisari), HEK293FT (Thermo Scientific), Vero cells (ATCC) and A549-ACE2 cells (gift from Olivier Schwartz) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Omega Scientific), penicillin/streptomycin (Gibco), non-essential amino acids (Gibco) and L-glutamine (Gibco) at 37C and 5% CO2.
    Huh7.5.1
    suggested: RRID:CVCL_E049)
    Software and Algorithms
    SentencesResources
    A total of 60 million mutagenized cells for each GeCKO sublibrary (A and B) were collected for genomic DNA extraction to assess the sgRNA representation of the starting population.
    GeCKO
    suggested: (Gecko, RRID:SCR_009001)
    The gene ontology enrichment of the individual screens was run on genes with MaGECK positive score <= 0.005 using the GO Biological Processes of the Molecular Signatures Database (MSigDB).
    GO Biological
    suggested: None
    The resulting network was clustered into subnetworks using the GLay Cytoscape plugin 68.
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.09.24.312298 on bioRxiv