Arginine Methylation Regulates SARS-CoV-2 Nucleocapsid Protein Function and Viral Replication

  1. Ting Cai
  2. Zhenbao Yu
  3. Zhen Wang
  4. Chen Liang
  5. Stéphane Richard

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Abstract

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5’-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.24.436822: (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
    1:2000); rabbit anti-SARS-CoV-2 N antibody (9103, Prosci, 1:2000); rabbit anti-SARS-CoV-2 S antibody (PA5-81795, Invitrogen, 1:1000);
    anti-SARS-CoV-2 N
    suggested: None
    anti-SARS-CoV-2 S
    suggested: None
    rabbit anti-TDRD3 antibody (Bethyl Laboratory,
    anti-TDRD3
    suggested: None
    mouse anti-SMN antibody (610646, BD Biosciences, 1:2000); rabbit anti-G3BP1 antibody (1F1, Rhône-Poulenc Rorer, 1:1000
    anti-SMN
    suggested: (BD Biosciences Cat# 610646, RRID:AB_397973)
    anti-G3BP1
    suggested: None
    Immunofluorescence were performed with the following antibodies: mouse anti-Flag monoclonal antibody (F1804, Sigma Aldrich, 1:500);
    anti-Flag
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    Alexa Fluor-conjugated goat anti-rabbit, goat anti-mouse secondary antibodies were from Invitrogen.
    anti-rabbit
    suggested: None
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture and transfection: HEK293 and VeroE6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and grown at 37°C with 5% CO2.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    HEK293 and Huh-7 cells were transfected with plasmid DNAs by standard calcium phosphate precipitation and Lipofectamine 3000 (Invitrogen), respectively.
    HEK293
    suggested: None
    Huh-7
    suggested: None
    Software and Algorithms
    SentencesResources
    rabbit anti-TDRD3 antibody (Bethyl Laboratory,
    Bethyl Laboratory
    suggested: (Bethyl, RRID:SCR_013554)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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 10.1101/2021.03.24.436822 on bioRxiv