ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1

  1. Boris Simonetti
  2. James L. Daly
  3. Lorena Simón-Gracia
  4. Katja Klein
  5. Saroja Weeratunga
  6. Carlos Antón-Plágaro
  7. Allan Tobi
  8. Lorna Hodgson
  9. Philip A. Lewis
  10. Kate J. Heesom
  11. Deborah K. Shoemark
  12. Andrew D. Davidson
  13. Brett M. Collins
  14. Tambet Teesalu
  15. Yohei Yamauchi
  16. Peter J. Cullen

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Abstract

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed “cargoes”) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans -Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

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  1. Version published to 10.1073/pnas.2201980119
  2. ScreenIT

    SciScore for 10.1101/2022.01.20.477115: (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
    Antibodies: Antibodies used in this study were: GFP (clones 7.1 and 13.1; 11814460001; Roche) (1:2000 for WB, 1:400 for IF), SNX1 (clone 51/SNX1; 611482; BD) (1:1000 for WB, 1:200 for IF), SNX2 (clone 13/SNX2; 5345661; BD) (1:1000 for WB, 1:200 for IF), SNX6 (clone d-5,365965; Santa Cruz Biotechnology, Inc.) (1:1000 for WB, 1:200 for IF), SNX5 (clone EPR14358; ab180520; Abcam) (WB 1:500
    GFP
    suggested: (Sigma-Aldrich Cat# 11814460001, RRID:AB_390913)
    SNX1
    suggested: (LSBio (LifeSpan Cat# LS-C109254-200, RRID:AB_10644956)
    SNX2
    suggested: (Santa Cruz Biotechnology Cat# sc-136072, RRID:AB_2192541)
    SNX6
    suggested: (Santa Cruz Biotechnology Cat# sc-365965, RRID:AB_10842310)
    clone d-5,365965; Santa Cruz Biotechnology, Inc.
    suggested: None
    SNX5
    suggested: None
    The cells were incubated with the mouse monoclonal anti-human NRP1 antibody (clone 3E7.1) at 4 °C for 30 min, washed with cold medium and, subsequently incubated with the AgNPs (0.3 nM in DMEM with 10% FBS) at 37 °C.
    anti-human NRP1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For GFP-based immunoprecipitations, HEK293T cells were transfected with GFP constructs using polyethylenimine (Sigma-Aldrich) and expression was allowed for 48 hours.
    HEK293T
    suggested: None
    For GFP-based immunoprecipitations, HEK-293T cells were lysed 48 h after transfection in immunoprecipitation buffer (50 mM Tris-HCl, 0.5% (v/v) NP-40, and Roche protease inhibitor cocktail) and subjected to GFP trap (ChromoTek).
    HEK-293T
    suggested: None
    Proteomics: HeLa cells expressing HRP-TGN46 were cultured for at least 6 doublings in three different isotopically labelled media compositions: R0K0 (light), R6K4 (medium), R10K8 (heavy) (Silantes).
    HeLa
    suggested: None
    PPC-1 cells (105 cells) were seeded onto noncoated coverslips (12 mm diameter, Marien-feld-Superior, Paul Marienfeld GmbH & Co.KG, Lauda-Königshofen, Germany) in a 24-well plate and cultured for 24 h.
    PPC-1
    suggested: ATCC Cat# HTB-190, RRID:CVCL_4778)
    Recombinant DNA
    SentencesResources
    To perform knockdowns in PPC-1 cells, pLKO.
    pLKO
    suggested: None
    1-puro-CMV-tGFP plasmids targeting SNX5 (5’-ACTATTACAATAGGATCAAAG-3’, 5’-CTGAGTATCTCGCTGTGTTTA-3’) and SNX6 (5’-AGTAAAGGATGTAGATGATTT-3’, 5’-GCCGAAACTTCCCAACAATTA-3’) (Sigma-Aldrich) were lentivirally transduced, and GFP-positive cells quantified as knockdowns.
    1-puro-CMV-tGFP
    suggested: None
    HeLa cells were transduced with lentiviruses, with constructs cloned into pXLG3 or pLVX vectors,to produce stably expressing cell lines.
    pXLG3
    suggested: None
    pLVX
    suggested: RRID:Addgene_174088)
    Recombinant Protein Expression and Purification: The expression plasmid of pGEX-4T-2 containing GST tagged SNX5 and SNX6 PX domain constructs were transformed into Escherichia coli BL 21 (DE3) cells and plated on lysogeny-broth (LB) agar plates supplemented with Ampicillin (0.1mg/mL).
    pGEX-4T-2
    suggested: RRID:Addgene_73945)
    Software and Algorithms
    SentencesResources
    Images were captured using Application Suite AF software (version 2.7.3.9723; Leica Microsystems) and then analyzed with the Volocity 6.3 software (PerkinElmer) or ImageJ.
    Volocity
    suggested: (Volocity 3D Image Analysis Software, RRID:SCR_002668)
    Statistical analyses were performed using GraphPad Prism 9 (LaJolla, CA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The raw data files were processed and quantified using Proteome Discoverer software v2.1
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    (Thermo Fisher Scientific) and searched against the UniProt Human database using the SEQUEST HT algorithm.
    UniProt
    suggested: (UniProtKB, RRID:SCR_004426)
    Gene ontology analysis was performed using the PANTHER classification system54 and Metascape55.
    PANTHER
    suggested: (PANTHER, RRID:SCR_004869)
    The colocalisation of the AgNPs with the different markers was quantified using the ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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 pages 39, 41, 42, 45 and 47. 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.

  3. Version published to 10.1101/2022.01.20.477115v1 on bioRxiv