Fibroblast-expressed LRRC15 suppresses SARS-CoV-2 infection and controls antiviral and antifibrotic transcriptional programs

  1. Lipin Loo
  2. Matthew A. Waller
  3. Cesar L. Moreno
  4. Alexander J. Cole
  5. Alberto Ospina Stella
  6. Oltin-Tiberiu Pop
  7. Ann-Kristin Jochum
  8. Omar Hasan Ali
  9. Christopher E. Denes
  10. Zina Hamoudi
  11. Felicity Chung
  12. Anupriya Aggarwal
  13. Jason K. K. Low
  14. Karishma Patel
  15. Rezwan Siddiquee
  16. Taeyoung Kang
  17. Suresh Mathivanan
  18. Joel P. Mackay
  19. Lukas Flatz
  20. Daniel Hesselson
  21. Stuart Turville
  22. G. Gregory Neely

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Abstract

Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here we use whole genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR -related cell surface receptor called leucine-rich repeat-containing protein 15 ( LRRC15 ). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe COVID-19 infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans . Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

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  1. Version published to 10.1101/2021.11.09.467981v2 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2021.11.09.467981: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variableIMR90 E6E7 (female) cells were a gift from Anthony Cesare (Children’s Medical Research Institute, Sydney,
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Cell lines have been authenticated.

    Table 2: Resources

    Antibodies
    SentencesResources
    At 72 hours post transduction the surface expression of ACE2 was measured by immunostaining the cells with anti-ACE2 monoclonal antibody (Thermo Fisher Scientific, MA5-32307)
    anti-ACE2
    suggested: (Thermo Fisher Scientific Cat# MA5-32307, RRID:AB_2809589)
    1 μg of anti-LRRC15 antibody (Abcam, EPR8188(2)) or rabbit IgG (Covance, CTL-4112) was added to 1 mg protein lysate and incubated at 4°C with rotation for 2.5 h before precipitation with protein G (ThermoFisher Scientific).
    anti-LRRC15
    suggested: None
    rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Experimental model and subject details: Cell culture: HEK293T cells (female; ATCC, CRL-3216, RRID: CVCL_0063) were cultured in Dulbecco’s Modified Eagle Medium (ThermoFisher Scientific, Cat #11995065) with 10% HyClone Fetal Bovine Serum (Cytiva, SH30084.03) and 1% Penicillin-Streptomycin (Gibco, 15140122) at 37°C, 5% CO2 and atmospheric oxygen.
    HEK293T
    detected: (CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Expi293F™ cells (female; ThermoFisher Scientific, A14527, RRID:CVCL_D615) were cultured in Expi293™ Expression Medium (ThermoFisher Scientific, A1435101) with 5% CO2 and atmospheric O2 at 37 °C for 24 h and then lowered to 32 °C for 72 h.
    Expi293F™
    detected: ( RRID:CVCL_D615)
    For individual sgRNA constructs, neat viral media was added to HEK293T-CRISPRa cells with Polybrene Infection / Transfection Reagent (Sigma-Aldrich) at a concentration of 8 ÎĽg/mL.
    HEK293T-CRISPRa
    suggested: None
    To transduce HEK293T cells, 10,000 cells per well were seeded in a 96 well tissue culture plate and virus supernatant added in a 2-fold dilution series.
    HEK293T
    suggested: None
    Lentiviral transductions were then performed on HEK293T-ACE2 cells to generate HEK293T-ACE2-TMPRSS2 cells.
    HEK293T-ACE2
    suggested: None
    For infection of cells with SARS-CoV-2 pseudovirus, WT HEK293T, HEK293T-ACE2 and HEK293T-ACE2-TMPRSS2 cells were transfected with cDNA for myc-DDK-tagged LRRC15 transcript 1, empty myc-DDK construct as a control plasmid.
    HEK293T-ACE2-TMPRSS2
    suggested: None
    Recombinant DNA
    SentencesResources
    Generation of CRISPR activation cell line: HEK293T cells were co-transfected with pPB-R1R2_EF1aVP64dCas9VP64_T2A_MS2p65HSF1-IRESbsdpA (Addgene #113341) and the Super PiggyBac Transposase Expression Vector (System Biosciences, PB210PA-1
    pPB-R1R2_EF1aVP64dCas9VP64_T2A_MS2p65HSF1-IRESbsdpA
    suggested: RRID:Addgene_113341)
    Oligonucleotides were annealed at 4°C for 16 h and pXPR-502 (Addgene #96923) was digested with Esp3I (ThermoFisher Scientific, ER0451) or BsmBI-v2 (New England Biolabs)
    pXPR-502
    suggested: None
    Lentivirus production and viral transduction: Lipofectamine 3000 Transfection Reagent (ThermoFisher Scientific) in Opti-MEM Medium (Gibco) was used to co-transfect HEK293T cells with psPAX2 (Addgene #12260), pCAG-VSVg (Addgene #35616) and either individual sgRNA constructs ligated into pXPR-502 (Addgene #96923) or pooled CRISPRa library (Human CRISPR activation pooled library set A, Addgene #92379) according to the manufacturer’s instructions.
    psPAX2
    suggested: RRID:Addgene_12260)
    pCAG-VSVg
    suggested: RRID:Addgene_35616)
    Briefly, ACE2 ORF was cloned into a 3rd generation lentiviral expression vector, pRRLsinPPT.CMV.GFP.WPRE (Follenzi et al., 2004) using Age1/BsrG1 cut sites, thus replacing GFP ORF with ACE2 to create a novel expression plasmid, herein referred to as ppt-ACE2.
    pRRLsinPPT.CMV.GFP.WPRE
    suggested: None
    Lentiviral particles expressing ACE2 were produced by co-transfecting ppt-ACE2, a 2nd generation lentiviral packaging construct psPAX2 and VSV-G plasmid pMD2.G (Addgene #12259) in HEK293T cells by using polyethylenimine as previously described (Aggarwal et al., 2012).
    VSV-G
    suggested: RRID:Addgene_138479)
    pMD2 . G
    suggested: None
    (synthetic gene fragment; IDT) was cloned into lentiviral expression vector pLVX-IRES-ZsGreen (Clontech) using EcoR1/XhoI restriction sites and lentiviral particles expressing TMPRSS2 were produced as described above.
    pLVX-IRES-ZsGreen
    suggested: None
    Immunoprecipitation: For SARS-CoV-2 spike pulldown, 2×107 HEK293T cells transfected with LRRC15-TurboGFP (transcript 1 and 2) or p□M1-EGFP (Addgene #19319) were incubated with 50 μg/mL spike hexapro for 30 min at 4°C with rotation.
    pâ–ˇM1-EGFP
    suggested: None
    Plasmid encoding the SARS-CoV-2 spike protein with an 18 amino acid truncation of the C-terminus was co-transfected into HEK293T cells with pBCKS(HIV-1SDmCMBeGFP-P2A-luc2pre-IIU), which permits equimolar expression of firefly luciferase and EGFP, and packaging plasmids pHCMVgagpolmllstwhv, pcDNA3.1tat101ml and pHCMVRevmlwhvpre.
    pBCKS(HIV-1SDmCMBeGFP-P2A-luc2pre-IIU
    suggested: None
    pHCMVgagpolmllstwhv
    suggested: None
    pcDNA3.1tat101ml
    suggested: None
    SARS-CoV-2 live virus infection assays: For assessing the inhibitory effect of native overexpression of LRRC15, HEK293T-ACE2-TMPRSS2 cells were transfected with myc-DDK-tagged LRRC15 transcript 1 plasmid (Origene, RC225990) for transient overexpression, with empty myc-DDK plasmid as a control plasmid.
    myc-DDK
    suggested: None
    1.25 ÎĽg empty TurboGFP plasmid were transfected for 0 ÎĽg LRRC15 cells, 0.3125 ÎĽg LRRC15 plasmid was mixed with 0.9375 ÎĽg empty TurboGFP plasmid for Lo LRRC15 cell transfection and 1.25 ÎĽg LRRC15-TurboGFP was transfected for Hi LRRC15 cells.
    TurboGFP
    suggested: None
    Software and Algorithms
    SentencesResources
    Conjugated spike proteins were loaded onto Bio-Rad BioGel P-30 Fine size exclusion purification resin column and eluted via gravity (Alexa Fluor™ 488) or centrifugation (Alexa Fluor™ 647).
    BioGel
    suggested: None
    LRR Tollkin Phylogenetic Tree: Protein sequences of LRR Tollkin family members (Dolan et al., 2007) were clustered using Clustal Omega (v1.2.2
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    Two single nucleus RNAseq datasets were downloaded from the Single Cell Portal (Broad Institute, SCP1052 and SCP1219) and one single cell RNAseq dataset from Gene Expression Omnibus (GSE158127)
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    Densitometry analysis of LRRC15, COL1A1 and ACTB were performed with ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Quantification and statistical analysis: SARS-CoV-2 spike glycoprotein titration experiments were analyzed on GraphPad Prism and fitted with non-linear regression (one site --specific binding) to identify maximal binding (Bmax) and dissociation constants (KD)
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    All density plots were generated using ggplot2.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 42. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

    Results from scite Reference Check: We found no unreliable references.

    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.

  5. Version published to 10.1101/2021.11.09.467981v1 on bioRxiv