Comparative host interactomes of the SARS-CoV-2 nonstructural protein 3 and human coronavirus homologs

  1. Katherine M. Almasy
  2. Jonathan P. Davies
  3. Lars Plate

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Abstract

Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, non-structural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) dataset for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance nuclear protein important for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.08.434440: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ) and THE anti-Strep II tag FITC (Genescript, A01736-100) antibodies.
    anti-Strep II tag FITC
    suggested: None
    A01736-100
    suggested: None
    , M2 anti-FLAG (Sigma-Aldrich, F1804), and anti-GAPDH (GeneTex, GTX627408) antibodies in 5% BSA.
    anti-FLAG
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    F1804
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    anti-GAPDH
    suggested: (GeneTex Cat# GTX627408, RRID:AB_11174761)
    Experimental Models: Cell Lines
    SentencesResources
    During the first IP for each construct, immunoprecipitation was confirmed by silver stain using a Pierce Silver Stain Kit (Thermo Fisher) and by western blotting with anti-FLAG antibody (Sigma-Aldrich, F1804) Nsp3.1-ST & ATF6-FT Co-immunoprecipitation: HEK293T-REx cells expressing doxycycline-inducible 6xHis-3xFLAG-HsATF6α were seeded in 15cm tissue culture dishes67.
    HEK293T-REx
    suggested: None
    Western blot and RT-qPCR analysis of UPR activation: HEK293T cells were transfected with nsp3.1-FT homologs or tdTomato (mock) in 6-well plates as previously described.
    HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    The amino acid sequences of full-length nsp3 for the remaining hCoV strains were aligned using ClustalOmega to the SARS-CoV fragments to determine the corresponding starting/ending positions for each fragment.
    ClustalOmega
    suggested: None
    Peptide IDs were filtered using Percolator with an FDR target of 0.01.
    Percolator
    suggested: (OMSSAPercolator, RRID:SCR_000287)
    Pairwise ratios between conditions were calculated in Proteome Discoverer based on total protein abundance, and ANOVA was performed on individual proteins to test for change in abundances and report adjusted P-values.
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    Geneset enrichment analysis, comparative heatmaps, and network plots: A gene ontology (GO) enrichment analysis for biological processes was conducted in EnrichR.
    EnrichR
    suggested: (Enrichr, RRID:SCR_001575)
    Network plots were generated in Cytoscape71; human protein interactions were validated based on the STRING database.
    STRING
    suggested: (STRING, RRID:SCR_005223)

    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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  2. Version published to 10.1101/2021.03.08.434440v1 on bioRxiv
  3. Version published to 10.1016/j.mcpro.2021.100120