SARS-CoV-2 protein Nsp1 alters actomyosin cytoskeleton and phenocopies arrhythmogenic cardiomyopathy-related PKP2 mutant

  1. Cristina Márquez-López
  2. Marta Roche-Molina
  3. Nieves García-Quintáns
  4. Silvia Sacristán
  5. David Siniscalco
  6. Andrés González-Guerra
  7. Emilio Camafeita
  8. Mariya Lytvyn
  9. María I. Guillen
  10. David Sanz-Rosa
  11. Daniel Martín-Pérez
  12. Cristina Sánchez-Ramos
  13. Ricardo García
  14. Juan A. Bernal

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Abstract

Mutations in desmosomal Plakophilin-2 (PKP2) are the most prevalent drivers of arrhythmogenic cardiomyopathy (ACM) and a common cause of sudden cardiac death in young athletes. However, partner proteins that elucidate PKP2 cellular mechanism to understand cardiac dysfunction in ACM are mostly unknown. Here we identify the actin-based motor proteins Myh9 and Myh10 as key PKP2 interactors, and demonstrate that the expression of the ACM-related PKP2 mutant R735X alters actin fiber organization and cell mechanical stiffness. We also show that SARS-CoV-2 Nsp1 protein acts similarly to this known pathogenic R735X mutant, altering the actomyosin component distribution on cardiac cells. Our data reveal that the viral Nsp1 hijacks PKP2 into the cytoplasm and mimics the effect of delocalized R735X mutant. These results demonstrate that cytoplasmic PKP2, wildtype or mutant, induces the collapse of the actomyosin network, since shRNA- PKP2 knockdown maintains the cell structure, validating a critical role of PKP2 localization in the regulation of actomyosin architecture. The fact that Nsp1 and PKP2 mutant R735X share similar phenotypes also suggests that direct SARS-CoV-2 heart infection could induce a transient ACM-like disease in COVID-19 patients, which may contribute to right ventricle dysfunction, observed in patients with poor survival prognosis.

Highlights

The specific cardiac isoform Plakophilin-2a (PKP2) interacts with Myh9 and Myh10.

PKP2 delocalization alters actomyosin cytoskeleton component organization. SARS-CoV-2 Nsp1 protein hijacks PKP2 from the desmosome into the soluble fraction where it is downregulated.

Viral Nsp1 collapses the actomyosin cytoskeleton and phenocopies the arrhythmogenic cardiomyopathy-related mutant R735X.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.14.296178: (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
    Then, cells were incubated with chicken α-GFP antibody (1:200, GFP-1010, Aves Labs), rabbit α-Myh9 antibody (1:200, GTX113236, GeneTex) and mouse α-Myh10 antibody (1:200, GTX634160, GeneTex) in 0.1% Triton X-100 PBS containing 10% NGS overnight at 4°C.
    α-GFP
    suggested: None
    GFP-1010
    suggested: (Antibodies Incorporated Cat# GFP-1010, RRID:AB_2307313)
    α-Myh9
    suggested: None
    α-Myh10
    suggested: None
    Proteins were separated from membrane and cytoplasmic extracts on 8% SDS-PAGE gels and then western blotted with antibodies against PKP2 (EB10841, Everest Biotech),
    PKP2
    suggested: None
    Secondary antibodies were anti-goat (ABIN2169607, Antibodies online) and anti-mouse (ABIN6699027,
    anti-goat ( ABIN2169607
    suggested: None
    Bound proteins were eluted with 30 μl loading buffer (10% SDS, 10 mM β-mercapto-ethanol, 20% glycerol, 200 mM Tris-HCl pH 6.8, 0.05% Bromophenolblue), heated for 15 minutes at 95°C, separated on 8% SDS-PAGE gels, and western blotted with antibodies against MYH9 (GTX633960, GeneTex), MYH10 (3404S, Cell Signaling Technology), and EGFP (632380, Clontech).
    MYH9
    suggested: (GeneTex Cat# GTX633960, RRID:AB_2888397)
    GTX633960
    suggested: (GeneTex Cat# GTX633960, RRID:AB_2888397)
    MYH10 ( 3404S , Cell Signaling Technology) ,
    suggested: None
    EGFP
    suggested: None
    Secondary antibodies were anti-mouse (ABIN6699027,
    anti-mouse
    suggested: None
    Antibodies online) and anti-rabbit (ABIN5563398,
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: The HEK293T (ATCC, CRL-3216) and HeLa (ATCC, CCL-2) cell lines were maintained in DMEM (GIBCO) supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mM L-glutamine.
    HEK293T
    suggested: None
    We analyzed 8 HL-1 cells expressing PKP2 and 5 cells each expressing the R735X and R735X-EGFP variants.
    HL-1
    suggested: None
    Immunostaining and imaging analysis: HeLa and HL-1 cell lines were fixed with 4% formaldehyde for 10 minutes, then permeabilized for 90 minutes at room temperature with 0.1% Triton X-100, and finally stained with Phalloidin-iFluor 594 Reagent (ab176757, Abcam).
    HeLa
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    For actomyosin components distribution analysis HL-1 EGFP stable cell lines were seeded at a density of 1×104/cm2 and cultured for 24 h.
    HL-1 EGFP
    suggested: None
    Software and Algorithms
    SentencesResources
    Maps were analyzed with in-house software written in Python.
    Python
    suggested: (IPython, RRID:SCR_001658)
    The MS/MS spectra were searched with the Sequest algorithm in Proteome Discoverer 1.4 (Thermo Scientific).
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    The percentage of actin filaments overlying the nucleus was calculated from the phalloidin intensity measured with Fiji software in all Z-stack images above the nucleus.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)

    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: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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 10.1101/2020.09.14.296178v1 on bioRxiv