SARS-CoV-2 desensitizes host cells to interferon through inhibition of the JAK-STAT pathway

  1. Da-Yuan Chen
  2. Nazimuddin Khan
  3. Brianna J. Close
  4. Raghuveera K. Goel
  5. Benjamin Blum
  6. Alexander H. Tavares
  7. Devin Kenney
  8. Hasahn L. Conway
  9. Jourdan K. Ewoldt
  10. Sebastian Kapell
  11. Vipul C. Chitalia
  12. Nicholas A. Crossland
  13. Christopher S. Chen
  14. Darrell N. Kotton
  15. Susan C. Baker
  16. John H. Connor
  17. Florian Douam
  18. Andrew Emili
  19. Mohsan Saeed

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Abstract

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.10.27.358259: (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
    Anti-SARS-CoV nucleocapsid (N) protein antibody (Rockland; #200-401-A50) was used for detection of SARS-CoV-2 N protein by IF.
    Anti-SARS-CoV nucleocapsid (N) protein
    suggested: (Rockland Cat# 200-401-A50, RRID:AB_828403)
    Anti-hACE2 antibodies included rabbit monoclonal antibody EPR4435(2) (Abcam; #ab108252) for Western blot and goat polyclonal antibody (R&D Systems; #AF933) for flow cytometry.
    Anti-hACE2
    suggested: None
    Goat IgG isotype control antibody was from Invitrogen (#02-6202) and Anti-VSV-G antibody from Kerafast (#EB0010).
    Goat IgG isotype control antibody
    suggested: None
    Goat IgG
    suggested: (Thermo Fisher Scientific Cat# 02-6202, RRID:AB_2532946)
    Anti-VSV-G
    suggested: (Kerafast Cat# EB0010, RRID:AB_2811223)
    #EB0010
    suggested: (Kerafast Cat# EB0010, RRID:AB_2811223)
    Antibodies for validation of our proteomics results and the follow-up studies included APOE (Proteintech; #18254-1-AP), AURKA (Bethyl Laboratories; #A300-071A), CHCHD2 (Proteintech; #19424-1-AP), JAK1 (Santa Cruz Biotechnology; #sc-277), LDLR (Bethyl Laboratories; #A304-417A), SERPINE1 (Novus Biologicals; NBP1-19773)
    CHCHD2
    suggested: (Proteintech Cat# 19424-1-AP, RRID:AB_10638907)
    JAK1
    suggested: (Santa Cruz Biotechnology Cat# sc-277, RRID:AB_631850)
    SERPINE1
    suggested: (Novus Cat# NBP1-19773, RRID:AB_2301656)
    Two ml of DMEM/10%FBS supplemented with anti VSV-G antibody was then added to each well to neutralize any residual input VSV.
    anti VSV-G
    suggested: None
    The cells were then permeabilized and incubated overnight at 4°C with anti-SARS-CoV Nucleocapsid antibody (1:2,000 dilution).
    anti-SARS-CoV
    suggested: None
    The cells were then washed 5 times with 1X PBS and stained with Alexa Fluor 568-conjugated goat anti-rabbit secondary antibody (1:1000 dilution) (Invitrogen; #A11008) in the dark at room temperature for 1h and counterstained with DAPI.
    anti-rabbit
    suggested: (Molecular Probes Cat# A-11008, RRID:AB_143165)
    Human ACE2 antibody or goat IgG isotype control was then added to the cells to obtain the final concentration of 5 μg/mL followed by 1h incubation on ice.
    ACE2
    suggested: None
    The cells were washed with FACS buffer and incubated for 30 minutes on ice in the dark with 1:400 dilution of Alexa Fluor 488 donkey anti-goat secondary antibody (Invitrogen; #A11055).
    anti-goat
    suggested: (Thermo Fisher Scientific Cat# A-11055, RRID:AB_2534102)
    Experimental Models: Cell Lines
    SentencesResources
    Human embryonic kidney HEK293T cells (ATCC; CRL-3216), human cervical carcinoma HeLa cells (ATCC; CCL-2), human colorectal adenocarcinoma HT-29 cells (ATCC; HTB-38), human hepatoblastoma HuH-6 cells (JCRB-0401), human hepatocellular carcinoma HuH-7 cells (JCRB-0403) and its derivative Huh-7.5 cells, human hepatocellular carcinoma HepG2 cells (ATCC; HB-8065), human lung anaplastic carcinoma Calu-6 cells (ATCC; HTB-56), human lung adenocarcinoma A549 cells (ATCC; CCL-185), human normal lung fibroblast MRC-5 cells (ATCC; CCL-171), human kidney papilloma HK-2 cells (ATCC; CRL-2190), human neuroblastoma SK-N-SH cells (ATCC; HTB-11), African green monkey kidney Vero E6 cells, and human muscle rhabdomyosarcoma RD cells (ATCC; CCL-136) were maintained in DMEM (Gibco; #11995-065) containing 10% FBS and 1X non-essential amino acids, human colorectal adenocarcinoma Caco-2 cells (ATCC; HTB-37) were maintained in the same medium but containing 20% FBS, human cardiomyocyte AC16 cells (Millipore; SCC109) in DMEM/F12 (Gibco; #11330-032) containing 12.5% FBS, human lung adenocarcinoma Calu-3 cells (ATCC; HTB-55) in MEM (Gibco; #11095-080) containing 10% FBS, and human umbilical vein endothelial HUV-EC-C cells (ATCC; CRL-1730) in RPMI 1640 medium (Gibco; #11875-093) containing 10% FBS.
    HEK293T
    suggested: None
    HeLa
    suggested: None
    HT-29
    suggested: None
    HuH-6
    suggested: JCRB Cat# JCRB0401, RRID:CVCL_1296)
    HuH-7
    suggested: RRID:CVCL_LG22)
    Huh-7.5
    suggested: RRID:CVCL_7927)
    HepG2
    suggested: ATCC Cat# HB-8065, RRID:CVCL_0027)
    Calu-6
    suggested: None
    A549
    suggested: None
    MRC-5
    suggested: None
    SK-N-SH
    suggested: None
    Caco-2
    suggested: None
    AC16
    suggested: None
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    HUV-EC-C
    suggested: ATCC Cat# CRL-1730, RRID:CVCL_2959)
    We then prepared the P2 working stock of the virus by infecting Vero E6 cells with the P1 stock at a multiplicity of infection (MOI) of 0.1 plaque forming units (PFU)/cell and harvesting the culture medium three days later.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Briefly, 293T cells were seeded into a 6-well plate at the density of 5×105 cells per well.
    293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Human embryonic kidney HEK293T cells (ATCC; CRL-3216), human cervical carcinoma HeLa cells (ATCC; CCL-2), human colorectal adenocarcinoma HT-29 cells (ATCC; HTB-38), human hepatoblastoma HuH-6 cells (JCRB-0401), human hepatocellular carcinoma HuH-7 cells (JCRB-0403) and its derivative Huh-7.5 cells, human hepatocellular carcinoma HepG2 cells (ATCC; HB-8065), human lung anaplastic carcinoma Calu-6 cells (ATCC; HTB-56), human lung adenocarcinoma A549 cells (ATCC; CCL-185), human normal lung fibroblast MRC-5 cells (ATCC; CCL-171), human kidney papilloma HK-2 cells (ATCC; CRL-2190), human neuroblastoma SK-N-SH cells (ATCC; HTB-11), African green monkey kidney Vero E6 cells, and human muscle rhabdomyosarcoma RD cells (ATCC; CCL-136) were maintained in DMEM (Gibco; #11995-065) containing 10% FBS and 1X non-essential amino acids, human colorectal adenocarcinoma Caco-2 cells (ATCC; HTB-37) were maintained in the same medium but containing 20% FBS, human cardiomyocyte AC16 cells (Millipore; SCC109) in DMEM/F12 (Gibco; #11330-032) containing 12.5% FBS, human lung adenocarcinoma Calu-3 cells (ATCC; HTB-55) in MEM (Gibco; #11095-080) containing 10% FBS, and human umbilical vein endothelial HUV-EC-C cells (ATCC; CRL-1730) in RPMI 1640 medium (Gibco; #11875-093) containing 10% FBS.
    Gibco
    suggested: None
    Anti-hACE2 antibodies included rabbit monoclonal antibody EPR4435(2) (Abcam; #ab108252) for Western blot and goat polyclonal antibody (R&D Systems; #AF933) for flow cytometry.
    R&D Systems
    suggested: None
    Antibodies for validation of our proteomics results and the follow-up studies included APOE (Proteintech; #18254-1-AP), AURKA (Bethyl Laboratories; #A300-071A), CHCHD2 (Proteintech; #19424-1-AP), JAK1 (Santa Cruz Biotechnology; #sc-277), LDLR (Bethyl Laboratories; #A304-417A), SERPINE1 (Novus Biologicals; NBP1-19773)
    Novus Biologicals
    suggested: (Novus Biologicals, RRID:SCR_004286)
    , SPARC (Proteintech; #15274-1-AP)
    SPARC
    suggested: (SPARC Project, RRID:SCR_017041)
    RT-qPCR was performed using Luna® Universal One-Step RT-qPCR kit (New England Biolabs; #E3005L).
    New England Biolabs
    suggested: (New England Biolabs, RRID:SCR_013517)
    Cells were resuspended in 1:50 dilution of human FC blocking solution (BioLegend; #422302) and incubated on ice for 10 min.
    BioLegend
    suggested: (BioLegend, RRID:SCR_001134)
    Data were collected using a BD LSR II flow cytometer and analyzed with FlowJo software (version 10).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Analysis of raw mass spectrometry data: All raw data were processed using MaxQuant (Version 1.6.7.0).
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)
    Data analysis and pathway enrichment: Bioinformatic analysis was performed using R: A language and environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org)
    Bioinformatic
    suggested: (QFAB Bioinformatics, RRID:SCR_012513)
    For functional enrichment of proteins based on clustering between the respective time points, Enrichr was used with the Reactome database of pathways.
    Enrichr
    suggested: (Enrichr, RRID:SCR_001575)
    On day 11, metabolic selection of hiPSC-CMs was started by growing them in a glucose-free RPMI 1640 medium (ThermoFisher Scientific; #11879020
    ThermoFisher Scientific
    suggested: None
    The intensity of protein bands was measured in the open source package, ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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:
    • 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 10.1101/2020.10.27.358259v1 on bioRxiv