ISG15-dependent activation of the sensor MDA5 is antagonized by the SARS-CoV-2 papain-like protease to evade host innate immunity

  1. GuanQun Liu
  2. Jung-Hyun Lee
  3. Zachary M. Parker
  4. Dhiraj Acharya
  5. Jessica J. Chiang
  6. Michiel van Gent
  7. William Riedl
  8. Meredith E. Davis-Gardner
  9. Effi Wies
  10. Cindy Chiang
  11. Michaela U. Gack

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Abstract

No abstract available

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  1. Version published to 10.1038/s41564-021-00884-1
  2. ScreenIT

    SciScore for 10.1101/2020.10.26.356048: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All work relating to SCoV2 live virus and SCoV2-RNA was conducted in the BSL-3 facility of the Cleveland Clinic Florida Research and Innovation Center in accordance with institutional biosafety committee (IBC) regulations.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and other reagents: Primary antibodies used in this study include anti-GST (1:5,000; Sigma-Aldrich), anti-V5 (1:5,000, R960-25; Novex), anti-FLAG (M2, 1:2,000; Sigma-Aldrich), anti-HA (1:3,000, HA-7; Sigma-Aldrich), anti-Phospho-IRF-3 (Ser396) (1:1,000, D6O1M; CST), anti-IRF3 (1:1,000, D6I4C; CST), anti-Phospho-STAT1 (Tyr701) (1:1,000, 58D6; CST), anti-IFIT1 (1:1,000, PA3-848; Invitrogen and 1:1,000, D2X9Z; CST), anti-IFIT2 (1:1,000; Proteintech), anti-ISG15 (1:500, F-9; Santa Cruz), anti-MAVS (1:1,000; CST), anti-RIG-I (1:2,000, Alme-1; Adipogen), anti-MDA5 (1:1,000, D74E4; CST), anti-Phospho-MDA5 (Ser88)7, anti-PP1α (1:2,000; Bethyl laboratories), anti-PP1γ (1:2,000; Bethyl laboratories), anti-USP18 (1:1000, D4E7; CST), anti-RSAD2 (1:1,000, D5T2X; CST), anti-PKR (1:1,000, D7F7; CST), anti-MX1 (1:1,000, D3W7I; CST), anti-IFITM3 (1:1,000, D8E8G; CST), anti-ISG20 (1:1,000, PA5-30073; Invitrogen), anti-ubiquitin (1:1,000, P4D1; Santa Cruz), anti-NS36, anti-PLpro (Nsp3) (1:1,000, GTX135589; GeneTex), anti-β-tubulin (1:1,000; CST), and anti-β-Actin (1:1,000, C4; Santa Cruz)
    anti-GST
    suggested: None
    anti-V5
    suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)
    anti-FLAG
    suggested: None
    anti-HA
    suggested: None
    anti-Phospho-IRF-3 ( Ser396 )
    suggested: (Cell Signaling Technology Cat# 29047, RRID:AB_2773013)
    anti-IRF3
    suggested: (Cell Signaling Technology Cat# 11904, RRID:AB_2722521)
    anti-Phospho-STAT1 ( Tyr701 )
    suggested: None
    anti-IFIT1
    suggested: (Antibodies-Online Cat# ABIN485037, RRID:AB_10847629)
    anti-IFIT2
    suggested: None
    anti-ISG15
    suggested: None
    anti-MAVS
    suggested: None
    anti-RIG-I
    suggested: None
    anti-Phospho-MDA5
    suggested: None
    anti-PP1α
    suggested: None
    anti-PP1γ
    suggested: None
    anti-USP18
    suggested: (Cell Signaling Technology Cat# 4813, RRID:AB_10614342)
    anti-RSAD2
    suggested: None
    anti-PKR
    suggested: (Cell Signaling Technology Cat# 12297, RRID:AB_2665515)
    anti-MX1
    suggested: (Cell Signaling Technology Cat# 37849, RRID:AB_2799122)
    anti-IFITM3
    suggested: (Cell Signaling Technology Cat# 59212, RRID:AB_2799561)
    anti-ISG20
    suggested: (Thermo Fisher Scientific Cat# PA5-30073, RRID:AB_2547547)
    anti-ubiquitin
    suggested: (Santa Cruz Biotechnology Cat# sc-8017, RRID:AB_628423)
    anti-NS36
    suggested: None
    anti-PLpro
    suggested: None
    Nsp3
    suggested: (GeneTex Cat# GTX135589, RRID:AB_2887501)
    anti-β-tubulin
    suggested: None
    anti-β-Actin
    suggested: None
    Monoclonal anti-IFNAR2 neutralizing antibody (1:250, MMHAR-2) was obtained from PBL Assay Science.
    anti-IFNAR2
    suggested: None
    Monoclonal anti-flavivirus E antibody (4G2) was purified from the mouse hybridoma cell line D1-4G2-4-15 (ATCC).
    Monoclonal anti-flavivirus E antibody
    suggested: (Absolute Antibody Cat# Ab00230-2.0, RRID:AB_2715504)
    Anti-mouse and anti-rabbit HRP-conjugated secondary antibodies (1:2,000) were purchased from CST.
    anti-rabbit HRP-conjugated secondary antibodies ( 1:2,000 )
    suggested: None
    Cell lysates were precleared with Protein G Dynabeads (Invitrogen) at 4°C for 2 h and then incubated with Protein G Dynabeads conjugated with the anti-MDA5 antibody or an IgG1 isotype control (G3A1; CST) at 4°C for 4 h.
    anti-MDA5
    suggested: None
    an IgG1
    suggested: None
    Cells were subsequently permeabilized with 1× BD Perm/Wash buffer (BD Biosciences) for 15 min and incubated with an anti-flavivirus E antibody (4G2; 1:100 in 1× BD Perm/Wash buffer) at 4°C for 30 min.
    anti-flavivirus E
    suggested: (Absolute Antibody Cat# Ab00230-2.0, RRID:AB_2715504)
    Cells were further washed three times with 1× BD Perm/Wash buffer and incubated with a goat anti-mouse Alexa Fluor 488-conjugated secondary antibody (#A10667, 1:500 in 1× BD Perm/Wash buffer; Invitrogen) at 4°C for 30 min in the dark.
    anti-mouse
    suggested: (Thermo Fisher Scientific Cat# A-10667, RRID:AB_2534057)
    Experimental Models: Cell Lines
    SentencesResources
    HEK293T-hACE2 and Vero-E6-hACE2 were a gift from Jae U.
    HEK293T-hACE2
    suggested: None
    A549-hACE2 were kindly provided by Benjamin R. tenOever (Icahn School of Medicine at Mount Sinai)4. HEK293T, HEK293, HeLa, MEFs, NHLFs, Vero, A549-hACE2, and BHK-21 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% (v/v)
    A549-hACE2
    suggested: None
    HEK293T-hACE2 and Vero-E6-hACE2 were maintained in DMEM containing 200 μg/mL hygromycin B and 2 μg/mL puromycin respectively.
    Vero-E6-hACE2
    suggested: None
    SVGA and HAP-1 cells were cultured in Eagle’s Minimum Essential Medium (MEM, Gibco) and Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco), respectively, supplemented with 10% FBS and 100 U/mL penicillin-streptomycin.
    HAP-1
    suggested: RRID:CVCL_5G07)
    C6/36 cells were cultured in MEM with 10% FBS and 100 U/mL penicillin-streptomycin.
    C6/36
    suggested: None
    Jung (Cleveland Clinic Lerner Research Center) and was propagated in Vero E6-hACE2 cells.
    Vero E6-hACE2
    suggested: None
    Additionally, MDA5-2CARD and its K23R/K43R mutant were subcloned into pcDNA3.1(-) harboring an N-terminal 3×FLAG tag between NheI and NotI. pCR3-FLAG-MV-V (strain Schwarz) was a gift from Karl-Klaus Conzelmann (LMU, Munich).
    MDA5-2CARD
    suggested: None
    Monoclonal anti-flavivirus E antibody (4G2) was purified from the mouse hybridoma cell line D1-4G2-4-15 (ATCC).
    D1-4G2-4-15
    suggested: BCRJ Cat# 0268, RRID:CVCL_J890)
    Briefly, HEK293T cells were transfected with GST or GST-MDA5-2CARD, and the cells were collected at 48 h post-transfection and lysed in Nonidet P-40 (NP-40) buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 1% (v/v) NP-40, 1 mM EDTA, and 1× protease inhibitor cocktail (Sigma)]
    HEK293T
    suggested: None
    Enzyme-linked immunosorbent assay (ELISA): Human or mouse IFN-β in the culture supernatants of NHLFs, HeLa, and MEFs was determined by ELISA using the VeriKine Human Interferon Beta ELISA Kit or VeriKine Mouse Interferon Beta ELISA Kit (PBL Assay Science) as previously described7. siRNA- and shRNA-mediated knockdown: Transient knockdown in NHLFs, HeLa, HAP-1, HEK293T, and HEK293 cells was performed using non-targeting or gene-specific siGENOME SMARTpool siRNAs (Dharmacon).
    HeLa
    suggested: None
    Briefly, HEK293T or MDA5 KO HEK293 cells were transfected with IFN-β luciferase reporter construct and β-galactosidase (β-gal) expressing pGK-β-gal, along with GST-MDA5-2CARD (WT or mutants) or FLAG-MDA5 (WT or mutants).
    MDA5 KO
    suggested: None
    Mock-RNA and SCoV2-RNA were produced by isolating total RNA from uninfected or SCoV2-infected (MOI 1 for 24 h) Vero-hACE2 cells.
    Vero-hACE2
    suggested: None
    The titers of ZIKV were determined by plaque assay on Vero cells as previously described6.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Flow cytometry: To quantify the percentage of DENV-infected cells, reconstituted HEK293 MDA5 KO cells were washed with PBS (Gibco) and fixed with 4% (v/v) formaldehyde in PBS at room temperature for 30 min.
    MDA5
    suggested: None
    Virus protection assay: The culture supernatants from mutant or WT EMCV-infected NHLFs or RIG-I KO HEK293 cells were UV-inactivated in a biosafety cabinet under a UV-C lamp (30W) at a dose of 5,000 μJ/cm2 for 15 min.
    HEK293
    suggested: None
    Complete inactivation of EMCV was confirmed by plaque assay on BHK-21 cells.
    BHK-21
    suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
    Software and Algorithms
    SentencesResources
    Briefly, HEK293T cells were transfected with GST or GST-MDA5-2CARD, and the cells were collected at 48 h post-transfection and lysed in Nonidet P-40 (NP-40) buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 1% (v/v) NP-40, 1 mM EDTA, and 1× protease inhibitor cocktail (Sigma)]
    Sigma
    suggested: (SigmaPlot, RRID:SCR_003210)
    Data analysis was performed using the FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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.

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  3. Version published to 10.1101/2020.10.26.356048 on bioRxiv