TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction

  1. Hataf Khan
  2. Helena Winstone
  3. Jose M. Jimenez-Guardeño
  4. Carl Graham
  5. Katie J. Doores
  6. Caroline Goujon
  7. David A. Matthews
  8. Andrew D. Davidson
  9. Suzannah J. Rihn
  10. Massimo Palmarini
  11. Stuart J. D. Neil
  12. Michael H. Malim

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Abstract

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike’s polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

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  1. Version published to 10.1371/journal.ppat.1009820
  2. ScreenIT

    SciScore for 10.1101/2021.07.23.453488: (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
    Proteins were transferred to a Hybond ECL membrane (Amersham biosciences) using a semi-dry transfer system (Biorad) and probed with primary antibodies: 1:1000 rabbit anti-ACE2 (Abcam, Ab108209) or 1:10000 mouse anti-tubulin (Sigma, T5168) and secondary antibodies: anti-mouse IgG IRdye 800CW (LI-COR Biosciences) or anti-rabbit IgG IRdye 800CW (LI-COR Biosciences)
    anti-ACE2
    suggested: None
    anti-tubulin
    suggested: None
    anti-mouse IgG
    suggested: None
    anti-rabbit IgG
    suggested: None
    Cells were blocked with 5% FCS in PBS (blocking buffer) for 30 min, and incubated with sheep anti-SARS-CoV-2 nucleocapsid antibody (1:10000) (86) in blocking buffer for 1 h.
    anti-SARS-CoV-2 nucleocapsid antibody (1:10000) (86
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Codon-optimised SARS-CoV-1, MERS-CoV, HCoV-229E and HCoV-NL63 Spike genes were synthesised in pcDNA3.1 from GeneArt.
    HCoV-229E
    suggested: None
    A549 and U87MG cells stably expressing ACE2 were generated by transduction with pMIGR1-blast-ACE2 vector and maintained with 10 μg/ml blasticidin selection.
    A549
    suggested: None
    U87MG
    suggested: None
    A549-ACE2 and U87MG-ACE2 cells stably expressing NCOA7 or E2crimson were generated by transduction with RRL.sin.cPPT.SFFV/IRES-puro.WPRE-containing vectors expressing NCOA7 or E2crimson and maintained with 2 μg/ml puromycin and 10 μg/ml balsticidin.
    U87MG-ACE2
    suggested: None
    A549-ACE2-NCOA7/E2crimson cells stably expressing TMPRSS2 were generated by transduction with RRL.sin.cPPT.SFFV/TMPRSS2(variant1).
    A549-ACE2-NCOA7/E2crimson
    suggested: None
    For CRISPR–Cas9-mediated gene disruption, A549-ACE2 cells stably expressing Cas9 were first generated by transduction with the LentiCas9-Blast vector followed by blasticidin selection at 10 μg/ml.
    A549-ACE2
    suggested: None
    Pseudotyped lentiviral vector production and infection: Pseudotyped lentiviral vectors were produced in a 10-cm dish seeded the day before with HEK293T cells.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    The infectious virus titre was determined by plaque assay in Vero-E6 cells.
    Vero-E6
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: NCOA7 variant 6 (NM_001199622.1) (encoding isoform 4 NP_001186551.1) or E2crimson cDNAs were inserted into pRRL.sin.cPPT.SFFV/IRES-puro.
    pRRL.sin.cPPT.SFFV/IRES-puro
    suggested: None
    Codon-optimised SARS-CoV-2 Spike in pcDNA3.1 and ACE2 expression constructs were kindly provided by Dr Nigel Temperton (82).
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    The ACE2 cDNA was inserted into pMIGR1-blast using Notl and Xhol to create
    pMIGR1-blast
    suggested: None
    The RNA guides were cloned into LentiGuide-Neo vector as described (31).
    LentiGuide-Neo
    suggested: RRID:Addgene_139449)
    A549 and U87MG cells stably expressing ACE2 were generated by transduction with pMIGR1-blast-ACE2 vector and maintained with 10 μg/ml blasticidin selection.
    pMIGR1-blast-ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    SARS-CoV-2 Spike P.1, B.1.1.1.7, B1.351 were synthesised in pcDNA3.1 from GENEWIZ.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Data were analysed with Graphpad Prism 9.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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.

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

    About SciScore

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  3. Version published to 10.1101/2021.07.23.453488 on bioRxiv