Ubiquitin ligase RIPLET mediates polyubiquitination of RIG-I and LGP2 and regulates the innate immune responses to SARS-CoV-2 infection

  1. Takahisa Kouwaki
  2. Tasuku Nishimura
  3. Guanming Wang
  4. Reiko Nakagawa
  5. Hiroyuki Oshiumi

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Abstract

RIG-I, a cytoplasmic viral RNA sensor, is crucial for innate antiviral immune responses; however, there are controversies about RIG-I’s regulatory mechanism by several ubiquitin ligases and LGP2. Our genetic study revealed that the RIPLET ubiquitin ligase was a general activating factor for RIG-I signaling, whereas another ubiquitin ligase, TRIM25, activated RIG-I in a cell-type-specific manner. These RIPLET and TRIM25 functions were modulated by accessory factors, such as ZCCH3C and NLRP12. Interestingly, we found an additional role of RIPLET in innate immune responses. RIPLET induced delayed polyubiquitination of LGP, resulting in the attenuation of excessive cytokine expression at the late phase. Moreover, RIPLET was involved in the innate immune responses against SARS-CoV-2 infection, a cause of the recent COVID-19 pandemic. Our data indicate that RIPLET fine-tunes innate immune responses via polyubiquitination of RIG-I and LGP2 against virus infection, including SARS-CoV-2.

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  1. Version published to 10.1101/2021.01.25.428042v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.01.25.428042: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationThe indicated number of cells were randomly chosen and observed under confocal microscopy (FV1200: Olympus) to determine the percentage of PLA-positive cells and the number of PLA signals in on cell.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-FLAG (1:150) Ab was added to the cell lysates, and then the lysate was incubated for 2 hours at 4 °C with rotation.
    Anti-FLAG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293FT and A549 cells were incubated in DMEM (high Glc) supplemented with 10% heat-inactivated FCS and penicillin-streptomycin solution.
    A549
    suggested: None
    HeLa cells were cultured in MEM supplemented with 10% heat-inactivated FCS and penicillin-streptomycin solution.
    HeLa
    suggested: None
    SeV was amplified using Vero cells, and the number of plaque-forming units was determined by a plaque assay.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Total RNA of VeroE6/TMPRSS2 cells infected with SARS-CoV-2 was isolated with TRIZOL, and cDNA was prepared using random primer.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Quantitative real-time PCR: We seeded 1 × 105 of HEK293 cells into 24-well plates.
    HEK293
    suggested: None
    GFP, LGP2 and LGP2 4KR ORFs were cloned into a FUIPW lentiviral vector and co-transfected with packaging plasmids into HEK293FT cells.
    HEK293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    Human ACE2 cDNA clone was synthesizes using HepG2 total RNA.
    HepG2
    suggested: CLS Cat# 300198/p2277_Hep-G2, RRID:CVCL_0027)
    The expression of ACE2 in HEK293 ACE2 cells was confirmed by RT-qPCR.
    HEK293 ACE2
    suggested: RRID:CVCL_DR94)
    Software and Algorithms
    SentencesResources
    The molecular masses of the resulting peptides were searched against the Uniprot Proteome Homo sapiens database (downloaded 2018.12.13) using the Mascot v2.6 program via Proteome Discoverer v2.2 (Themo Fisher Scientific) with the false discovery rate (FDR) set at 0.01.
    Mascot
    suggested: (Mascot, RRID:SCR_014322)
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    Statistical significance (p-value) was determined using a two-tailed Student’s t-test, one-way ANOVA, or two-way ANOVA in Prism v7.0a (GraphPad Software) and MS-Excel (Microsoft Corp) software. *p < 0.05.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 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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  3. Version published to 10.1101/2021.01.25.428042v1 on bioRxiv