LRRC15 mediates an accessory interaction with the SARS-CoV-2 spike protein

  1. Jarrod Shilts
  2. Thomas W. M. Crozier
  3. Ana Teixeira-Silva
  4. Ildar Gabaev
  5. Pehuén Pereyra Gerber
  6. Edward J. D. Greenwood
  7. Samuel James Watson
  8. Brian M. Ortmann
  9. Christian M. Gawden-Bone
  10. Tekle Pauzaite
  11. Markus Hoffmann
  12. James A. Nathan
  13. Stefan Pöhlmann
  14. Nicholas J. Matheson
  15. Paul J. Lehner
  16. Gavin J. Wright

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Abstract

The interactions between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and human host factors enable the virus to propagate infections that lead to Coronavirus Disease 2019 (COVID-19). The spike protein is the largest structural component of the virus and mediates interactions essential for infection, including with the primary angiotensin-converting enzyme 2 (ACE2) receptor. We performed two independent cell-based systematic screens to determine whether there are additional proteins by which the spike protein of SARS-CoV-2 can interact with human cells. We discovered that in addition to ACE2, expression of LRRC15 also causes spike protein binding. This interaction is distinct from other known spike attachment mechanisms such as heparan sulfates or lectin receptors. Measurements of orthologous coronavirus spike proteins implied the interaction was functionally restricted to SARS-CoV-2 by accessibility. We localized the interaction to the C-terminus of the S1 domain and showed that LRRC15 shares recognition of the ACE2 receptor binding domain. From analyzing proteomics and single-cell transcriptomics, we identify LRRC15 expression as being common in human lung vasculature cells and fibroblasts. Levels of LRRC15 were greatly elevated by inflammatory signals in the lungs of COVID-19 patients. Although infection assays demonstrated that LRRC15 alone is not sufficient to permit viral entry, we present evidence that it can modulate infection of human cells. This unexpected interaction merits further investigation to determine how SARS-CoV-2 exploits host LRRC15 and whether it could account for any of the distinctive features of COVID-19.

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  1. Version published to 10.1371/journal.pbio.3001959
  2. ScreenIT

    SciScore for 10.1101/2021.09.25.461776: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Following three more HBS-T washes, 0.2 μg/mL of a secondary anti-mouse IgG antibody linked to alkaline phosphatase (Sigma #A9316) was incubated for 30 minutes.
    a secondary anti-mouse IgG antibody
    suggested: (Molecular Probes Cat# A-31553, RRID:AB_221604)
    anti-mouse IgG
    suggested: (Sigma-Aldrich Cat# A9316, RRID:AB_258446)
    In the case of fluorescent-conjugated tetramer staining, cells were finally resuspended in 1% BSA in PBS, while in the case of Fc fusion protein staining, cells were stained with secondary antibody conjugated to AlexaFluor 647 against human IgG-gamma1 (Jackson #109-605-006), washed, and then resuspended.
    human IgG-gamma1
    suggested: None
    Coverslips were then incubated in TBS + 2% BSA containing the indicated dilution of primary rabbit anti-human LRRC15 antibody (ab150376) for 1 h at room temperature, washed three times in TBS + 2% BSA, incubated for 30 minutes in TBS + 2% BSA containing highly cross-adsorbed goat anti-rabbit Alexa Fluor 555 conjugated secondary antibody (2 μg/mL) and DAPI (0.2 μg/mL), then washed three times in TBS and once in distilled water prior to mounting slides with Prolong Glass (Thermo Fisher) followed by overnight curing.
    anti-human LRRC15
    suggested: (Creative Biomart Cat# CPBT-54314RH, RRID:AB_11420140)
    anti-rabbit
    suggested: None
    Blocked membranes were incubated with primary antibody (LRRC15 [LSBio aa393-422], ACE2 [Abcam Ab108252], GAPDH [GeneTex GTX627408]) in 5% milk +PBST (PBS + 0.2% (v/v) Tween-20) at 4°C overnight, then incubated with peroxidase (HRP)-conjugated secondary antibodies (Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) [Jackson ImmunoResearch 111-035-144], Peroxidase AffiniPure
    ACE2
    suggested: None
    GAPDH
    suggested: (GeneTex Cat# GTX627408, RRID:AB_11174761)
    Anti-Rabbit IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 111-035-144, RRID:AB_2307391)
    Experimental Models: Cell Lines
    SentencesResources
    HEK293 cell culturing: For both binding assays and the production of recombinant proteins, suspension HEK293-E cells (Durocher et al., 2002) were grown in Freestyle media (Gibco #12338018) supplemented with 1% (m/v
    HEK293
    suggested: None
    SARS-CoV-2 S1-Fc protein was purified from serum-free supernatant of transfected 293T cells by Protein A affinity chromatography using hiTrap MabSelect PrismA columns (GE Healthcare) and the Äkta-Pure liquid chromatography system.
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    HEK-293T and hTERT RPE-1 cells were grown in Dulbecco Modified Eagle Media (DMEM) supplemented with 10% fetal calf serum (FCS).
    HEK-293T
    suggested: None
    VSV-G lentiviruses were produced by transfection of HEK293T cells with a lentiviral expression vector and packaging vectors pCMVΔR8.91 and pMD.G at a DNA ratio of 3:2:1 using TranslT-293 (Mirus) following the manufacturers recommendation.
    HEK293T
    suggested: None
    Production of SARS-CoV-2 viral stocks: A total of 1 μg of pCCI-4K-SARS-CoV-2-ZsGreen plasmid DNA and 3 μL of Lipofectamine LTX with 3 μL of PLUS reagent in 100 μL optiMEM were used to transfect BHK-21 cells (ECACC) in 6-well plates.
    BHK-21
    suggested: None
    SARS-CoV-2 microscopy infection assays: RPE-1 or CaLu-3 cell lines were seeded into CellCarrier-96 Black optically clear bottom plates (Perkin Elmer) at a density of either ×1×104 cells/well 24 h prior to infection or ×5×104 cells/well 72 h prior to infection respectively.
    CaLu-3
    suggested: None
    Recombinant DNA
    SentencesResources
    Individual CRISPRa guides were cloned into pCRISPRia-v2 (Addgene #84832).
    pCRISPRia-v2
    suggested: RRID:Addgene_84832)
    ACE2 and LRRC15 cDNAs were cloned into pHRSIN-cSGW vectors expressing puromycin, blasticidin or hygromycin resistance cassettes driven by a pGK promoter.
    pHRSIN-cSGW
    suggested: None
    VSV-G lentiviruses were produced by transfection of HEK293T cells with a lentiviral expression vector and packaging vectors pCMVΔR8.91 and pMD.G at a DNA ratio of 3:2:1 using TranslT-293 (Mirus) following the manufacturers recommendation.
    pCMVΔR8.91
    suggested: None
    pMD.G
    suggested: None
    Production of SARS-CoV-2 viral stocks: A total of 1 μg of pCCI-4K-SARS-CoV-2-ZsGreen plasmid DNA and 3 μL of Lipofectamine LTX with 3 μL of PLUS reagent in 100 μL optiMEM were used to transfect BHK-21 cells (ECACC) in 6-well plates.
    pCCI-4K-SARS-CoV-2-ZsGreen
    suggested: None
    Software and Algorithms
    SentencesResources
    For data analysis, single-end 35 bp reads were trimmed down to the variable sgRNA segment using FASTX-Toolkit and aligned to an index of all sequences in the library using Bowtie 2.
    Bowtie
    suggested: (Bowtie, RRID:SCR_005476)
    Phylogenetics analysis: Homologs for LRRC15 were identified by BLAST searches against the human LRRC15 amino acid sequence, filtering for species reported to be potential SARS-CoV-2 hosts (Liu et al., 2021; Mahdy et al., 2020).
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Multiple sequence alignments were done by the Clustal Omega algorithm using default parameters (Madeira et al., 2019).
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    Phylogenetic trees were drawn using Jalview (v. 2.11.1.4) from average distance calculations based on the BLOSUM62 substitution matrix.
    Jalview
    suggested: (Jalview, RRID:SCR_006459)
    The data were visualized with the Scanpy package (v. 1.4.5) in Python (v. 3.7.4)
    Python
    suggested: (IPython, RRID:SCR_001658)
    These data were exported as .fcs files and analysed in FlowJo.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: Thank you for sharing your data.

    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 36. 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

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  3. Version published to 10.1101/2021.09.25.461776v1 on bioRxiv