The Host Interactome of Spike Expands the Tropism of SARS-CoV-2

  1. Casimir Bamberger
  2. Sandra Pankow
  3. Salvador Martínez-Bartolomé
  4. Jolene Diedrich
  5. Robin Park
  6. John Yates

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

The SARS-CoV-2 virus causes severe acute respiratory syndrome (COVID-19) and has rapidly created a global pandemic. Patients that survive may face a slow recovery with long lasting side effects that can afflict different organs. SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor Angiotensin Converting Enzyme 2 (ACE2) which binds to spike protein trimers on the surface of SARS-CoV-2 virions. However, SARS-CoV-2 can spread to other tissues even though they are negative for ACE2. To gain insight into the molecular constituents that might influence SARS-CoV-2 tropism, we determined which additional host factors engage with the viral spike protein in disease-relevant human bronchial epithelial cells (16HBEo − ). We found that spike recruited the extracellular proteins laminin and thrombospondin and was retained in the endoplasmatic reticulum (ER) by the proteins DJB11 and FBX2 which support re-folding or degradation of nascent proteins in the ER. Because emerging mutations of the spike protein potentially impact the virus tropism, we compared the interactome of D614 spike with that of the rapidly spreading G614 mutated spike. More D614 than G614 spike associated with the proteins UGGT1, calnexin, HSP7A and GRP78/BiP which ensure glycosylation and folding of proteins in the ER. In contrast to G614 spike, D614 spike was endoproteolytically cleaved, and the N-terminal S1 domain was degraded in the ER even though C-terminal ‘S2 only’ proteoforms remained present. D614 spike also bound more laminin than G614 spike, which suggested that extracellular laminins may function as co-factor for an alternative, ‘S2 only’ dependent virus entry. Because the host interactome determines whether an infection is productive, we developed a novel proteome-based cell type set enrichment analysis (pCtSEA). With pCtSEA we determined that the host interactome of the spike protein may extend the tropism of SARS-CoV-2 beyond mucous epithelia to several different cell types, including macrophages and epithelial cells in the nephron. An ‘S2 only’ dependent, alternative infection of additional cell types with SARS-CoV-2 may impact vaccination strategies and may provide a molecular explanation for a severe or prolonged progression of disease in select COVID-19 patients.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.02.16.431318: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationFurthermore, each cell type enrichment score is differentiated from a population of enrichment scores (suprema) that is obtained in an iterative process in which cell type labels are randomly permutated, yielding an empirical p-value that indicates the significance of enrichment.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Immunofluorescence detection and protein co-localization: 48 hours post transfection cells were washed with 1x PBS, fixed in 4% paraformaldehyde/1x PBS for 15 min at RT, permeabilized with 0.1 % Triton X-100/1xPBS for 10 min at RT, washed three times with 1x PBS and incubated with 5 % horse serum/1xPBS for 1 h at 20 °C before incubating with the following antibodies (Cell Signaling Laboratories): anti-calnexin (clone C5C9), anti-BiP (clone C50B12), anti-Hsp90a (clone D1A7), anti-ZO1 (clone D7D12), anti-ZO2 (2847), anti-ZO3 (D57G7), anti-claudin 1 (D5H1D), anti-CD2AP (2135), anti-afadin (clone D1Y3Z) or Alexa Fluor 488 anti-DYKDDDDK (flag) tag monoclonal antibody (L5, Life Technologies).
    anti-calnexin
    suggested: (Thermo Fisher Scientific Cat# MA3-027-A488, RRID:AB_2662809)
    anti-BiP
    suggested: (Cell Signaling Technology Cat# 3177, RRID:AB_2119845)
    anti-Hsp90a
    suggested: None
    anti-ZO3 (D57G7), anti-claudin 1
    suggested: None
    anti-CD2AP
    suggested: (Cell Signaling Technology Cat# 2135, RRID:AB_2228922)
    anti-afadin
    suggested: (Cell Signaling Technology Cat# 13531, RRID:AB_2798249)
    anti-DYKDDDDK
    suggested: None
    Proteins were separated on NuPAGE 4-12% Bis-Tris gels (Life technologies) before transferring proteins onto Nitrocellulose membranes and incubating with anti-BiP (C50B12, Cell Signaling Laboratories (CSL)), anti-ZO1 (D7D12, CSL), anti-ZO2 (2847, CSL) or Alexa Fluor 488 conjugated anti-DYKDDDDK Tag monoclonal antibody (L5, Sigma) in 5 % BSA, 1x PBS, 0.05 % Tween-20.
    anti-BiP (C50B12
    suggested: (Cell Signaling Technology Cat# 3177, RRID:AB_2119845)
    anti-ZO1
    suggested: None
    anti-ZO2
    suggested: None
    Signals were detected either using Radiance Q ECL reagent (Azure Biosystems) after washing and incubation with HRP coupled anti-rabbit antibodies (CSL) or imaged directly using an Azure 600 Western blot imaging system.
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture, transfection and transduction: HEK293T cells were grown at 37 °C and 5 % CO2 in DMEM supplemented with 10% tetracycline-free fetal bovine serum and 1% Penicillin-Streptomycin (Invitrogen).
    HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Fragment ion spectra were searched with Prolucid 66 with the forward and reversed human UniProt database v.07-01-2020, and filtered with DTASelect 67 to a false discovery rate of < 1 %.
    UniProt
    suggested: (UniProtKB, RRID:SCR_004426)
    Western blot images were quantified in Fiji/ImageJ.
    Fiji/ImageJ
    suggested: None

    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.

  2. Version published to 10.1101/2021.02.16.431318v1 on bioRxiv