The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

  1. Matthias M. Zimmer
  2. Anuja Kibe
  3. Ulfert Rand
  4. Lukas Pekarek
  5. Liqing Ye
  6. Stefan Buck
  7. Redmond P. Smyth
  8. Luka Cicin-Sain
  9. Neva Caliskan

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Abstract

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

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  1. Version published to 10.1038/s41467-021-27431-0
  2. ScreenIT

    SciScore for 10.1101/2021.05.31.445667: (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
    1 mg of cell lysate was cleared with protein A magnetic beads to remove non-specific interactions (S1425S, NEB) and incubated overnight with anti-ZAP antibody (Proteintech 16820-1-AP) or anti-IgG from rabbit as a control (Cell Signaling, a gift from Dr. Mathias Munschauer, HIRI-HZI).
    anti-ZAP
    suggested: None
    anti-IgG
    suggested: None
    After transfer using Trans-Blot (Bio-Rad), nitrocellulose membranes were developed using the following primary antibodies: anti-His-tag (ab18184), anti-DDDDK (ab49763), anti-ALFA (FluoTag®-X2 anti-ALFA AlexaFluor 647), anti-ZC3HAV1 (Proteintech 16820-1-AP).
    anti-His-tag
    suggested: None
    anti-ALFA
    suggested: (Antibodies-Online Cat# ABIN125862, RRID:AB_11186338)
    anti-ZC3HAV1
    suggested: (Proteintech Cat# 16820-1-AP, RRID:AB_2728733)
    The following secondary antibodies were used: IRDye® 800CW Goat anti-rabbit and IRDye® 680RD Donkey anti-Mouse (both LI-COR).
    anti-rabbit
    suggested: (LI-COR Biosciences Cat# 926-68073, RRID:AB_10954442)
    The nitrocellulose membranes were developed using anti-DDDDK primary (Abcam ab49763) and IRDye® 680RD donkey anti-mouse secondary antibody (LI-COR).
    anti-DDDDK
    suggested: (Abcam Cat# ab49763, RRID:AB_869428)
    anti-mouse
    suggested: None
    For the experiments, optical tweezers (OT) constructs were mixed with 4 μl of polystyrene beads coated with antibodies against digoxigenin (AD beads, 0.1% v/v suspension, Ø 1.76 μm, Spherotech), 10 μl of assay buffer (20 mM HEPES, pH 7.6, 300 mM KCl, 5 mM MgCl2, 5 mM DTT and 0.05% Tween) and 1 μl of RNase inhibitor.
    digoxigenin
    suggested: (Millipore Cat# AQ300D, RRID:AB_11212694)
    Experimental Models: Cell Lines
    SentencesResources
    Calu-3 cells (ATCC HTB-55) were cultured in MEM (Sigma) supplemented with 10% FBS.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    The supernatant was used to transduce naïve Huh7 cells in the presence of 10 μg/ml polybrene (Merck Millipore).
    Huh7
    suggested: None
    The virus was raised for two passages on Caco-2 cells (HZI Braunschweig).
    Caco-2
    suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
    To study the effect of ZAP-S on SARS-CoV-2 infection, Huh-7 cells were employed.
    Huh-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Cells were infected with 200000 pfu/ml, corresponding to an MOI of 0.03 at 24h post-infection, cell culture supernatants were collected and titrated by plaque assay on Vero E6 cells (ATCC CRL-1586).
    Vero E6
    suggested: None
    Polysome profiling analysis: A plasmid expressing ZAP-S N-terminally tagged with a His-tag was transfected into HEK293 cells using PEI, as described above.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    To check endogenous ZAP-S expression, HEK cells were transfected with a plasmid containing the same backbone and His-tag.
    HEK
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Recombinant DNA
    SentencesResources
    TFRC (NM_003234.4), ZAP (NM_024625.4) and ZNF346 (NM_012279.4) were placed in frame with the coding sequence for ECFP in pFlp-Bac-to-Mam (gift from Dr.
    pFlp-Bac-to-Mam
    suggested: None
    Protein-coding sequences were introduced by Golden Gate Assembly using AarI cut sites 69. pET-SUMO-GFP (gift from Prof. Utz Fischer, Julius-Maximilians-University, Würzburg, Germany) was used as the parental vectors for protein overexpression in E. coli.
    pET-SUMO-GFP
    suggested: None
    Optical tweezers constructs were based on the wild type SARS-CoV-2 frameshifting site (nucleotides 13475-13541) cloned into the plasmid pMZ_lambda_OT, which encodes for the optical tweezer handle sequences (2Kb each) flanking the RNA structure (130 nt).
    pMZ_lambda_OT
    suggested: None
    VSV-G envelope pseudo-typed lentivirus for the generation of stable cell lines was produced by co-transfection of each transfer plasmid with pCMVdR 8.91 71 and pCMV-VSV-G (gift from Prof. Weinberg, Addgene plasmid # 8454 72).
    pCMV-VSV-G
    suggested: RRID:Addgene_8454)
    RNAs were labeled at the 3’ end using pCp-Cy5 (Cytidine-5’-phosphate-3’-(
    pCp-Cy5
    suggested: None
    Software and Algorithms
    SentencesResources
    Flow cytometry data were analyzed with FlowJo software (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Bands corresponding to the –1 or 0-frame products, 58 kDa and 33 kDa respectively, on western blots of in vitro translations were quantified densitometrically using ImageJ software 74.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Graphs were plotted using GraphPad Prism 8.4.3 software. Microscopy: HEK293 cells were cultured on glass slides and transfected as described above.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The results were plotted using Prism 8.0.2 (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    All data are available in the main text, the supplementary materials as well as in Mendeley Data: DOI: 10.17632/c7rbxb86k2.1
    Mendeley
    suggested: (Mendeley Data, RRID:SCR_002750)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT811Trial number did not resolve on clinicaltrials.gov. Is the number correct?NA

    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.

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

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  3. Version published to 10.21203/rs.3.rs-563757/v1 on Research Square
  4. Version published to 10.1101/2021.05.31.445667v1 on bioRxiv