ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of liver cells

  1. Xinyi Yang
  2. Yuqi Zhu
  3. Xiaying Zhao
  4. Jun Liu
  5. Jiangna Xun
  6. Songhua Yuan
  7. Jun Chen
  8. Hanyu Pan
  9. Jinlong Yang
  10. Jing Wang
  11. Zhimin Liang
  12. Xiaoting Shen
  13. Yue Liang
  14. Qinru Lin
  15. Huitong Liang
  16. Min Li
  17. Hongzhou Lu
  18. Huanzhang Zhu

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Abstract

Backgroud & Aims

Currently, the COVID-19 pandemic, caused by SARS-CoV-2 infection, represents a serious public health problem worldwide. Although it has been shown that ACE2 serves as the main receptor for SARS-CoV-2 entry into host cells, studies have shown that ACE2 is expressed at extremely low levels in various tissues, especially in some organs where virus particles have been found, such as the heart and liver. Therefore, these organs potentially express additional SARS-CoV-2 receptors that have not yet been discovered.

Methods & Results

Here, by a genome-wide CRISPR-Cas9 activation library screening, we found that ASGR1 promoted SARS-CoV-2 infection of 293T cells. In Huh-7 and HepG2 cell lines, simultaneous knock out of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary liver parenchymal cells, both of which hardly express ACE2, SARS-CoV-2 could successfully establish an infection. After treatment with ASGR1 antibody, the infection rate significantly reduced. This suggests that SARS-CoV-2 infects liver cells mainly through an ASGR1-dependent mechanism. Finally, we also found that the soluble ASGR1 could not only prevent the SARS-CoV-2 pseudovirus, which binds to the ASGR1 receptors, from infecting host liver cells, but also had a protective effect on those expressing ACE2, indicating that administration of soluble ASGR1 protein may represent a new treatment approach.

Conclusions

Colletively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of liver cells.

Lay Summary

We show that ASGR1 is a candidate receptor for SARS-CoV-2 to infect liver cells.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2022.01.15.476426: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibody and reagents: The following antibodies were used throughout this study: from ABclonal(Wuhan, China), anti-FLAG (AE063), anti-HA (AE036), anti-ASGR1 (A13279), anti- β -Actin (AC026).
    anti-FLAG
    suggested: None
    anti-HA
    suggested: None
    anti-ASGR1
    suggested: (ABclonal Cat# A13279, RRID:AB_2760131)
    anti- β
    suggested: None
    AC026
    suggested: (ABclonal Cat# AC026, RRID:AB_2768234)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: 293T, HeLa, HepG-2, Huh-7, H1299, A549, BEAS-2B, cells were were cultured in DMEM (Gibco, C11995500BT) with 10% fetal bovine serum (FBS) (ExCell Bio, FSP500) and 1% penicillin/streptomycin (P/S) (Gibco, 15140-122) in a 37 ° C incubator containing 5% CO2.
    293T
    suggested: None
    HeLa
    suggested: None
    HepG-2
    suggested: None
    H1299
    suggested: None
    A549
    suggested: None
    BEAS-2B
    suggested: None
    CaCo-2 cells were cultured in 1640 (Gibco, C11875500BT) and supplemented with 10% FBS (ExCell Bio, FSP500), and 1%P/S in a 37 °C incubator containing 5% CO2.
    CaCo-2
    suggested: None
    Huh-7 or 293T cells were infected with lentivirus at an MOI of 1 and then selected using 2 μg/ml puromycin for 14 days for knockout or overexpression.
    Huh-7
    suggested: None
    For production of pseudoviruses, HEK293T/17 cells were seeded into 10cm dishes one day before transfection.
    HEK293T/17
    suggested: None
    Protein purification: The ASGR1 protein was produced in HEK 293T cells.
    HEK 293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    Recombinant DNA
    SentencesResources
    The core plasmid, pLenti.
    pLenti
    suggested: RRID:Addgene_125133)
    When the cells reached 80% confluence, plasmid and PEI were added into the Opti-MDM (Gibco), mixed evenly, and left standing for 20min. pLenti.GFP.NLuc, psPAX2 and the S protein vector were co-transfected into HEK293T/17 cells to produce the pseudoviruses.
    pLenti.GFP.NLuc
    suggested: None
    psPAX2
    suggested: RRID:Addgene_12260)
    Software and Algorithms
    SentencesResources
    FlowJo software (FlowJo LLC, Ashland, OR) was used to perform the flow cytometry analysis.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Paired samples t-tests were performed with use of SPSS version 13.0 (SPSS Inc., Chicago), and statistical significance was indicated at *P < 0.05, **P < 0.01 or ***P < 0.001.
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    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 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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2022.01.15.476426v1 on bioRxiv