Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV

  1. Alexey Stukalov
  2. Virginie Girault
  3. Vincent Grass
  4. Ozge Karayel
  5. Valter Bergant
  6. Christian Urban
  7. Darya A. Haas
  8. Yiqi Huang
  9. Lila Oubraham
  10. Anqi Wang
  11. M. Sabri Hamad
  12. Antonio Piras
  13. Fynn M. Hansen
  14. Maria C. Tanzer
  15. Igor Paron
  16. Luca Zinzula
  17. Thomas Engleitner
  18. Maria Reinecke
  19. Teresa M. Lavacca
  20. Rosina Ehmann
  21. Roman Wölfel
  22. Jörg Jores
  23. Bernhard Kuster
  24. Ulrike Protzer
  25. Roland Rad
  26. John Ziebuhr
  27. Volker Thiel
  28. Pietro Scaturro
  29. Matthias Mann
  30. Andreas Pichlmair

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Abstract

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  1. Version published to 10.1038/s41586-021-03493-4
  2. Version published to 10.1101/2020.06.17.156455v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.06.17.156455: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    For detection of protein abundance by western blotting, HA-HRP (Sigma-Aldrich), ACTB-HRP (Santa Cruz), ATM, MAP1LC3B, MAVS, HSPA1A, TGFβ and SQSTM1, phospho-JNK (T183/Y185), JNK, phospho-p38 (T180/Y182), p38 (Cell Signaling), SARS-CoV-2 (Sino Biological) antibodies were used.
    ATM
    suggested: None
    HSPA1A
    suggested: None
    SQSTM1
    suggested: None
    phospho-JNK (T183/Y185)
    suggested: (R and D Systems Cat# AF1205, RRID:AB_2140857)
    JNK
    suggested: (Thermo Fisher Scientific Cat# 85-86195-11, RRID:AB_2574775)
    phospho-p38 (T180/Y182)
    suggested: (R and D Systems Cat# MAB8691, RRID:AB_10890618)
    p38
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and reagents: HEK293T, A549, Vero E6 and HEK293R1 cells and their respective culturing conditions were described previously22.
    HEK293R1
    suggested: RRID:CVCL_9831)
    A549 cells were transduced twice, and ACE2-expressing A549 (ACE2-A549) cells were selected with puromycin.
    ACE2-A549
    suggested: None
    Virus strains, stock preparation, plaque assay and in vitro infection: SARS-CoV-2-MUC-IMB-1 and SARS-CoV-2-GFP strains20 were produced by infecting Vero E6 cells cultured in DMEM medium (10% FCS, 100 ug/ml Streptomycin, 100 IU/ml Penicillin) for 2 days (MOI 0,01).
    Vero E6
    suggested: None
    A549-ACE2 cells were infected with SARS-CoV-2-MUC-IMB-1 strain (MOI 3) for the subsequent experiments.
    A549-ACE2
    suggested: None
    Affinity purification mass spectrometric analyses of SARS-COV-2, SARS-COV and HCoV protein expressing A549 cells: For the determination of SARS-COV-2, SARS-COV and partial HCoV interactomes, four replicate affinity purifications were performed for each HA-tagged viral protein.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Ingenuity knowledge base was used as a reference dataset, only experimentally observed findings were used for confidence filtering, additionally human species and A549-ATCC cell line filters were set.
    A549-ATCC
    suggested: None
    Co-immunoprecipitation and western blot analysis: HEK293T cells were transfected with pWPI plasmid encoding single HA-tagged viral proteins, alone or together with pTO-SII-HA expressing host factor of interest.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Total amounts of IFN-α/β in cell supernatants were measured by using 293T cells stably expressing the firefly luciferase gene under the control of the mouse Mx1 promoter (Mx1-luc reporter cells)47.
    293T
    suggested: None
    Briefly, HEK293RI cells were seeded, transfected with pCAGGS-flag-RIG-I plus viral protein constructs and stimulated as described above.
    HEK293RI
    suggested: RRID:CVCL_9831)
    Experimental Models: Organisms/Strains
    SentencesResources
    Virus strains, stock preparation, plaque assay and in vitro infection: SARS-CoV-2-MUC-IMB-1 and SARS-CoV-2-GFP strains20 were produced by infecting Vero E6 cells cultured in DMEM medium (10% FCS, 100 ug/ml Streptomycin, 100 IU/ml Penicillin) for 2 days (MOI 0,01).
    SARS-CoV-2-GFP
    suggested: None
    Software and Algorithms
    SentencesResources
    Expression constructs for C-terminal HA tagged viral ORFs were synthesised (Twist Bioscience and BioCat) and cloned into pWPI vector as described previously23 with the following modifications: starting ATG codon was added, internal canonical splicing sites were replaced with synonymous mutations and C-terminal HA-tag, followed by amber stop codon, was added to individual viral open reading frames.
    BioCat
    suggested: (BioCAT, RRID:SCR_001440)
    To acquire MS data, the data-independent acquisition (DIA) scan mode operated by the XCalibur software (Thermo Fisher) was used.
    XCalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    Spectra were searched against forward and reverse sequences of the reviewed human proteome including isoforms (UniprotKB, release 10.2019) and SARS-COV-2, SARS-COV and HCoV proteins by the built-in Andromeda search engine31.
    UniprotKB
    suggested: (UniProtKB, RRID:SCR_004426)
    Bioinformatic analysis: Unless otherwise specified, the bioinformatic analysis was done in R (version 3.6), Julia (version 1.4) and Python (version 3.8) using a collection of in-house scripts (available upon request).
    Python
    suggested: (IPython, RRID:SCR_001658)
    Statistical analysis of DDA total proteome, phosphoproteome and ubiquitinome data 6 and 24 hours post SARS-CoV-2 infection of A549-ACE2 cells: The output of MaxQuant was analyzed with Perseus (version 1.6.14.0)36 and visualized with R (version 3.6.0) and RStudio (version 1.2.1335).
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)
    Perseus
    suggested: (Perseus, RRID:SCR_015753)
    RStudio
    suggested: (RStudio, RRID:SCR_000432)
    Annotation of detected protein groups, phosphosites and ubiquitination sites with GOBP, -MF, -CC, KEGG, Pfam, GSEA, Keywords and Corum as well as PhosphoSitePlus kinase-substrate relations and regulatory sites (version May 1st 2020)37 was performed in Perseus.
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    Pfam
    suggested: (Pfam, RRID:SCR_004726)
    Corum
    suggested: (CORUM, RRID:SCR_002254)
    Transcripts with low mean normalized count that were flagged by the independent filtering procedure of DESeq2 were removed and those with absolute apeglm shrunk log2 fold change > 0.5 and the p-value < 0.05 were considered differentially expressed in distinct conditions.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Gene Set Enrichment Analysis: We have used Gene Ontology, Reactome and other EnrichmentMap gene sets of human proteins41 as well as protein complexes annotations from IntAct Complex Portal (version 2019.11)42 and CORUM (version 2019)43.
    EnrichmentMap
    suggested: (EnrichmentMap, RRID:SCR_016052)
    Transcription factor – target gene set libraries from ENCODE were used45.
    ENCODE
    suggested: (Encode, RRID:SCR_015482)
    Transcriptome, proteome, ubiquitinome and phosphoproteome changes along with unchanged transcripts/proteins/sites were submitted to the core ingenuity pathway analysis (IPA) (www.ingenuity.com).
    ingenuity pathway analysis
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)

    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

    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.

  4. ScreenIT

    SciScore for 10.1101/2020.06.17.156455: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    To evaluate the consequences of these interactions on cellular proteostasis, we proceeded with the total proteome analysis of A549 cells expressing the 54 individual viral proteins (Figure 1a, d; Supplementary Table 3).
    A549
    suggested: None
    (c-f) Co-precipitation experiments in HEK 293T cells showing a specific enrichment of (c) endogenous MAVS co-precipitated with c-term HA-tagged ORF7b of SARS-CoV-2 and SARS-CoV (negative controls: SARS-CoV-2 ORF6-HA, ORF7a-HA), (d) ORF7bHA of SARS-CoV-2 and SARS-CoV co-precipitated with SII-HA-UNC93B1 (control precipitation: SII-HA-RSAD2), (e) endogenous HSPA1A co-precipitated with N-HA of SARS-CoV-2 and SARSCoV (control: SARS-CoV-2 ORF6-HA) and (f) endogenous TGFβ with ORF8-HA of SARS-CoV-2 vs ORF8-HA, ORF8a-HA, ORF8b-HA of SARS-CoV or ORF9b-HA of SARS-CoV-2. (g, h) Differential enrichment of proteins in (g) NSP2 and (h) ORF8 of SARS-CoV-2 (x-axis) vs SARS-CoV (y-axis) AP-MS experiments.
    HEK 293T
    suggested: None
    (T183/Y185, MAPK8) in SARS-CoV-2 infected A549-ACE2 cells.
    A549-ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    (d) Gene Ontology Biological Processes enriched among the cellular proteins that are up- (red arrow) or down(blue arrow) regulated upon overexpression of individual viral proteins.
    Ontology Biological
    suggested: None
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.17.156455. this version posted June 17, 2020.
    bioRxiv
    suggested: (bioRxiv, SCR_003933)

    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 OddPub: Thank you for sharing your data.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  5. Version published to 10.1101/2020.06.17.156455v1 on bioRxiv