Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities

  1. Thomas Kruse
  2. Caroline Benz
  3. Dimitriya H. Garvanska
  4. Richard Lindqvist
  5. Filip Mihalic
  6. Fabian Coscia
  7. Raviteja Inturi
  8. Ahmed Sayadi
  9. Leandro Simonetti
  10. Emma Nilsson
  11. Muhammad Ali
  12. Johanna Kliche
  13. Ainhoa Moliner Morro
  14. Andreas Mund
  15. Eva Andersson
  16. Gerald McInerney
  17. Matthias Mann
  18. Per Jemth
  19. Norman E. Davey
  20. Anna K. Överby
  21. Jakob Nilsson
  22. Ylva Ivarsson

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Abstract

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.

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  1. Version published to 10.1038/s41467-021-26498-z
  2. ScreenIT

    SciScore for 10.1101/2021.04.19.440086: (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.
    RandomizationNumber of cells highly infected (strong N staining) was counted in 12 random fields (magnification 20x) and % of cells expressing stress granule (G3BP1) signals in these were calculated.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Virus was detected using primary monoclonal rabbit antibodies directed against SARS-CoV-2 nucleocapsid (Sino Biological Inc., 40143-R001), and secondary anti-rabbit HRP conjugated antibodies (1:2000, Thermo Fisher Scientific).
    SARS-CoV-2 nucleocapsid ( Sino Biological Inc . , 40143-R001) ,
    suggested: None
    anti-rabbit HRP
    suggested: None
    Antibodies: The following antibodies were used at the indicated dilutions: c-Myc (1:1000, Santa Cruz Biotechnology, sc-40), rabbit anti-G3BP1 (WB; 1:1000
    c-Myc
    suggested: (Santa Cruz Biotechnology Cat# sc-40 AC, RRID:AB_2857941)
    WB
    suggested: None
    mouse APC-conjugated antibody directed against dsRNA J2 (IF 1:200, Scicons, 10010500) mouse anti-3xFlag M2 (WB 1:25000, Sigma,
    anti-3xFlag
    suggested: None
    F1804), rabbit anti-Tubulin (WB 1:4000, Abcam, ab6046)
    anti-Tubulin
    suggested: None
    donkey anti-rabbit IgG (Invitrogen), goat anti-rabbit HRP conjugated antibody (WB 1:2000 or 1:5000, Thermo Fisher Scientific, 31460)
    donkey anti-rabbit IgG
    suggested: None
    anti-rabbit IgG
    suggested: (Thermo Fisher Scientific Cat# 31460, RRID:AB_228341)
    goat anti-mouse HRP conjugated antibody (WB 1:5000, Thermo Fisher Scientific, 31430)
    goat anti-mouse HRP
    suggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307)
    anti-mouse HRP
    suggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307)
    Thereafter, cells are incubated with primary antibodies against SARS-CoV-2 nucleocapsid ((1:500) Sino Biological Inc., 40143-R001) and G3BP1 ((1:500
    SARS-CoV-2 nucleocapsid
    suggested: None
    G3BP1
    suggested: None
    ) Abcam, ab56574) followed by incubation with conjugated secondary antibodies anti-rabbit Alexa555 and anti-mouse Alexa488 (1:500, Thermo Fisher Scientific).
    anti-rabbit
    suggested: None
    anti-mouse
    suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)
    Proteins were separated with SDS-PAGE and Western blot analysis was performed using antibodies against SARS CoV-2 nucleocapsid, 3xFlag M2 (Sigma, F1804) and tubulin (Abcam, ab6046)
    antibodies against SARS CoV-2 nucleocapsid ,
    suggested: None
    F1804
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    tubulin ( Abcam ,
    suggested: (Abcam Cat# ab106042, RRID:AB_10898490)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture, virus and reagents: HeLa and HEK293 cells were maintained in DMEM GlutaMAX containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS (all from Thermo Fisher Scientific)
    HEK293
    suggested: None
    Lentivirus production, transductions and virus infection: Lenti-X 293T cells (Takara bio) grown in a 100 mm plate format were co-transfected with 4.5 μg psPAX2 (Didier Trono lab, Addgene plasmid #12260), 500 ng pMD2.
    293T
    suggested: None
    For transductions VeroE6 or HEK cells were seeded into greiner CELLSTAR® 96-well plates or 6-well plates (VWR) containing lentivirus in DMEM containing 2 % FBS and 1 μg/mL polybrene, and incubated for 72 h.
    HEK
    suggested: None
    Immunoprecipitation of SARS-CoV/SARS-CoV2/MERS-CoV N proteins: Two 15 cm3 dishes were seeded with HeLa cells at 20% confluency.
    HeLa
    suggested: None
    VeroE6 cells were seeded in 8-well chamber slides (Sarstedts) and infected with SARS CoV-2 for 6 h.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Envelope 3xFlag (pGBW-m4252867 was a gift from Ginkgo Bioworks & Benjie Chen (Addgene plasmid # 153626; http://n2t.net/addgene:153626; RRID: Addgene_153626) and pcDNA5/FRT/TO Myc SARS CoV2 N and N 2A was transfected into HEK293T cells using GeneJuice (Novagen, Darmstadt, Germany) following the manufacturer’s protocol (12 μg of DNA/sample in total).
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Recombinant DNA
    SentencesResources
    To generate the YFP-G3BPi inhibitor, the double FGDF motif from Semliki Forest virus nsP3 (RTTFRNKLPFTFGDFDEHEVDALASGITFGDFDDVL) or a control inhibitor (RTTFRNKLPATAGDFDEHEVDALASGITAGDADDVL) were fused to the C terminus of YFP by cloning the DNA encoding this into the pcDNA5/FRT/TO vector (Invitrogen).
    pcDNA5/FRT/TO
    suggested: RRID:Addgene_19445)
    Lentivirus production, transductions and virus infection: Lenti-X 293T cells (Takara bio) grown in a 100 mm plate format were co-transfected with 4.5 μg psPAX2 (Didier Trono lab, Addgene plasmid #12260), 500 ng pMD2.
    psPAX2
    suggested: RRID:Addgene_12260)
    pMD2
    suggested: None
    To generate transfer plasmids, four copies of inhibitory peptide (three copies for the Semilikiforest peptide) or control peptides with the binding motifs mutated were fused to C terminus of EGFP and cloned to pLJM1-EGFP vector (David Sabatini lab, Addgene plasmid #19319).
    pLJM1-EGFP
    suggested: RRID:Addgene_19319)
    Assembly assay: pcDNA3.1 expression plasmids coding for the structural proteins of SARS CoV-2, Spike (S D614), Membrane (M) (was a gift from Jeremy Luban
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    Envelope 3xFlag (pGBW-m4252867 was a gift from Ginkgo Bioworks & Benjie Chen (Addgene plasmid # 153626; http://n2t.net/addgene:153626; RRID: Addgene_153626) and pcDNA5/FRT/TO Myc SARS CoV2 N and N 2A was transfected into HEK293T cells using GeneJuice (Novagen, Darmstadt, Germany) following the manufacturer’s protocol (12 μg of DNA/sample in total).
    pGBW-m4252867
    suggested: None
    Software and Algorithms
    SentencesResources
    The sample was analyzed using Illumina MiSeq v3 run, 1×150bp read setup, 20% PhiX by the NGS-NGI SciLifeLab facility.
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    Results were processed using in-house custom Python scripts described previsouly22.
    Python
    suggested: (IPython, RRID:SCR_001658)
    To assess the quality of RiboVD library we analyzed the coverage percentage of the phage library over the library design using the NGS results of non-challenged naive library phage aliquotes. 95.50% of the peptide sequences designed to be in the library were confirmed by the NGS analysis of the naïve phage library.
    NGS
    suggested: (PM4NGS, RRID:SCR_019164)
    The peptide data was combined with available information on SG localized proteins from mass spectroscopy of purified mammalian stress granules27, 52, 59 and from other studies based on the information listed in the HIPPIE database.
    HIPPIE
    suggested: (HIPPIE, RRID:SCR_014651)
    Peptides were from GeneCust (France) with a purity of over 95%.
    GeneCust
    suggested: None
    Bioinformatic analysis: Proteomics data analysis was performed with Perseus65 and within the R environment (https://www.r-project.org/).
    https://www.r-project.org/
    suggested: (R Project for Statistical Computing, RRID:SCR_001905)
    MaxQuant output tables were filtered for ‘Reverse’, ‘Only identified by site modification’, and ‘Potential contaminants’ before data analysis.
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)

    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: 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

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  3. Version published to 10.1101/2021.04.19.440086v1 on bioRxiv