Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics

  1. Li Wang
  2. Adil Muneer
  3. Ling Xie
  4. Feng Zhang
  5. Bing Wu
  6. Liu Mei
  7. Erik M Lenarcic
  8. Emerald Hillary Feng
  9. Juan Song
  10. Yan Xiong
  11. Xufen Yu
  12. Charles Wang
  13. Ciprian Gheorghe
  14. Karina Torralba
  15. Jeanette Gowen Cook
  16. Yisong Y. Wan
  17. Nathaniel John Moorman
  18. Hongjun Song
  19. Jian Jin
  20. Xian Chen

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Abstract

Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.14.382416: (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
    Antibodies against G9a (07-551) and H3K9me2 (07-441) were from Millipore; Antibodies against EIF4E(11149-1-AP), EIF3B(10319-1-AP), HNRNPA2B1(14813-1-AP),METTL3(15073-1-AP), YTHDF2(24744-1-AP),PD-L1 (66248-1-lg), CDC20(10252-1-AP), CDCA7(15249-1-AP), BIRC5(10508-1-AP), TPX2(11741-1-AP), TOP2A(24641-1-AP), NUSAP1(12024-1-AP), SPP1(22952-1-AP), SQSTM1(18420-1-AP), ACTIN(60008-1-lg) were from Proteintech.
    G9a
    suggested: (Millipore Cat# 07-551, RRID:AB_310709)
    H3K9me2
    suggested: None
    EIF4E(11149-1-AP
    suggested: None
    EIF3B(10319-1-AP
    suggested: None
    HNRNPA2B1
    suggested: (Proteintech Cat# 14813-1-AP, RRID:AB_2279638)
    METTL3
    suggested: (Proteintech Cat# 15073-1-AP, RRID:AB_2142033)
    YTHDF2
    suggested: (Proteintech Cat# 24744-1-AP, RRID:AB_2687435)
    PD-L1 ( 66248-1-lg) , CDC20
    suggested: None
    CDCA7
    suggested: (Proteintech Cat# 15249-1-AP, RRID:AB_2878119)
    BIRC5
    suggested: None
    TPX2
    suggested: (Proteintech Cat# 11741-1-AP, RRID:AB_2208895)
    TOP2A(24641-1-AP
    suggested: None
    NUSAP1
    suggested: (Proteintech Cat# 12024-1-AP, RRID:AB_2157789)
    SPP1
    suggested: None
    22952-1-AP
    suggested: (Proteintech Cat# 22952-1-AP, RRID:AB_2783651)
    SQSTM1
    suggested: None
    Antibody against p-p65 (S536)(3033) was from Cell Signaling.
    p-p65
    suggested: (Millipore Cat# PP65-K, RRID:AB_1603731)
    S536
    suggested: None
    Antibody against Brg1 (H-88) (sc-10768x) was from Santa Cruz
    Brg1
    suggested: None
    Anti-HA (clone HA-7) and anti-flag M2 (clone M2) antibodies were from Sigma. m6A RNA methylation quantification kit was from Abcam(ab185912).
    Anti-HA
    suggested: None
    anti-flag
    suggested: None
    Ten percent of 150 ng mRNA was used as Input mRNA, and the remainder was incubated with 3 ug anti-m6A polyclonal antibody (Synaptic Systems; 202003) which was preconjugated to Dynabeads Protein A (Thermo Fisher; 10001D) in 500uL IP buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Igepal CA-630) for 2 hours at 4°C.
    anti-m6A
    suggested: (Synaptic Systems Cat# 202 003, RRID:AB_2279214)
    Experimental Models: Cell Lines
    SentencesResources
    HEK 293 stable TLR4-MD2-CD14 cell line was also purchased from InvivoGen.
    HEK 293
    suggested: None
    Raw264.7 and THP1 cells were purchased from ATCC (Manassas,VA).
    THP1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
    For MeRIP-Seq, THP-1 cells were first incubated in the presence of 60 nM PMA overnight to differentiate into macrophages followed by 48 h resting in PMA-free medium.
    THP-1
    suggested: None
    Transfection and CRISPR Knockout: 293TLR4-MD2-CD14 cells were seeded in 6-well plates for 24 h before transfection, and the constructs were transfected or co-transfected into MCF7 cells using reagent jetPRIME (Polyplus).
    293TLR4-MD2-CD14
    suggested: None
    MCF7
    suggested: NCI-DTP Cat# MCF7, RRID:CVCL_0031)
    In brief, a total of 10 μg of plasmid, including target plasmid, pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) with a ratio of 10:5:9, was co-transfected into 293T cells with jetPRIME™.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    MDA-MB-231 cells at 60-80% confluency were incubated with the virus containing media for 24-48 hours, and then subjected to 1.0 μg/mL puromycin selection.
    MDA-MB-231
    suggested: None
    METTL3 KO, G9a KO, and control Raw 264.7 cells were seeded in 6-well plates with indicated treatments followed by 50□μg/mL Mitomycin C treatment for 30□min at 37□°C.
    Raw 264.7
    suggested: None
    Software and Algorithms
    SentencesResources
    Protein quantitation was performed using TMT reporter intensity found in MaxQuant and a one-sample t-test statistics on three technical replicates was used with a p-value of 5% to report statistically significant protein abundance fold-changes.
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)
    Analysis of Functional Category and Networks: The biological processes and molecular functions of the G9a-interacting proteins were categorized by IPA (http://www.ingenuity.com/), DAVID (http://david.abcc.ncifcrf.gov/), and STRING (http://string-db.org/).
    http://www.ingenuity.com/
    suggested: (Ingenuity Pathways Knowledge Base, RRID:SCR_008117)
    DAVID
    suggested: (DAVID, RRID:SCR_001881)
    STRING
    suggested: (STRING, RRID:SCR_005223)
    Ten percent of 150 ng mRNA was used as Input mRNA, and the remainder was incubated with 3 ug anti-m6A polyclonal antibody (Synaptic Systems; 202003) which was preconjugated to Dynabeads Protein A (Thermo Fisher; 10001D) in 500uL IP buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Igepal CA-630) for 2 hours at 4°C.
    Synaptic Systems
    suggested: (Synaptic Systems, RRID:SCR_013612)
    Thermo Fisher
    suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
    Then the Input and m6A-IP reads were mapped to human genome version hg38 by STAR v.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    The turnover rates (kdeg/ksyn), curve maxima (Mo/Ho), offsets (γ) and goodness-of-fit statistics (SSE, RMSE, rsquared, adjrsquared) were obtained for each protein using a nonlinear least square (NLS) method in MATLAB (vR2017b).
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)

    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 10.1101/2020.11.14.382416v1 on bioRxiv