Cap-independent translation and a precisely located RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response

  1. Boris Slobodin
  2. Urmila Sehrawat
  3. Anastasia Lev
  4. Daniel Hayat
  5. Binyamin Zuckerman
  6. Davide Fraticelli
  7. Ariel Ogran
  8. Amir Ben-Shmuel
  9. Elad Bar-David
  10. Haim Levy
  11. Igor Ulitsky
  12. Rivka Dikstein

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Abstract

Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represses translation, and induces mRNA degradation, while the host elicits an anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-mediated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5′UTR of SARS-CoV-2 drives cap-independent translation and promotes expression of NSP1 in an eIF4E-independent and Torin1-resistant manner. Upon expression, NSP1 further enhances cap-independent translation. However, the sub-genomic 5′UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin1. We conclude that the combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5′UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

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  1. Version published to 10.1093/nar/gkac615
  2. ScreenIT

    SciScore for 10.1101/2021.08.18.456855: (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 AuthenticationAuthentication: To monitor growth kinetics, the cells were seeded on 96-well plates, treated as indicated and subjected to CellTiter-Glo luminescent cell viability assay (Promega) according to the manufacturer’s instructions.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies used: anti-puromycin antibodies (Millipore Cat#MABE343),
    anti-puromycin
    suggested: (Millipore Cat# MABE343, RRID:AB_2566826)
    Experimental Models: Cell Lines
    SentencesResources
    mRNA library preparation for MARS-seq: Total RNA was isolated from HEK293T cells using Bio Tri RNA reagent (Bio-lab) and mRNA was captured using Oligo d(T)25 magnetic Beads (NEB) according to manufacturer’s protocol.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Biological resources: Cultured cell lines: MCF7 and HEK293 cells were from ATCC, MRC5 and Vero E6 cells were generously provided by Zvi Livneh and Yosef Shaul, respectively (WIS, Israel).
    MCF7
    suggested: None
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Virus stocks were propagated (four passages) and titered on Vero E6 cells (Vero E6, ATCC® CRL-1586™).
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    Reagents: DNA constructs: To create HA-tagged NSP1 lacking viral 5’UTR for expression in cell culture (pCRUZ-HA-NSP1), NSP1 was obtained from the Forchheimer plasmid bank (WIS, Israel) and cloned into pCRUZ-HA (sc-5045) plasmid (Santa Cruz Biotechnology) using restriction-free cloning and primers #1,2 (here and on, see Table S1 for primers’ sequences).
    pCRUZ-HA-NSP1
    suggested: None
    pCRUZ-HA
    suggested: None
    To create HA-tagged NSP1 with the viral 5’UTR for expression in cell culture (pcDNA3.1-TSS-5’UTR-HA-NSP1), HA-NSP1 was PCR-amplified from the pCRUZ-HA-NSP1 plasmid using primers #5,6 and introduced immediately after the transcription start site of the pcDNA3.1(-) plasmid using restriction-free cloning.
    pcDNA3.1-TSS-5’UTR-HA-NSP1
    suggested: None
    reporter gene bearing Hisx6 tag was amplified using primers #18,19 and cloned between HindIII-KpnI sites of pcDNA5/FRT/TO plasmid.
    pcDNA5/FRT/TO
    suggested: RRID:Addgene_19445)
    The resulting product was used in a PCR to amplify pcDNA3.1(-) plasmid as template.
    pcDNA3.1 ( - )
    suggested: None
    Nsp1 protein purification for in vitro assay: The pET28 plasmid encoding for His-tagged NSP1 was transformed by heat shock into BL-21 E.coli and a single grown colony was inoculated into 5ml of LB supplemented with Kanamycin (50μg/ml) and incubated shaking at 37°C.
    pET28
    suggested: RRID:Addgene_21766)
    Software and Algorithms
    SentencesResources
    mRNA library preparation for MARS-seq: Total RNA was isolated from HEK293T cells using Bio Tri RNA reagent (Bio-lab) and mRNA was captured using Oligo d(T)25 magnetic Beads (NEB) according to manufacturer’s protocol.
    Oligo
    suggested: (oligo, RRID:SCR_015729)
    After treatment with ECL reagent (Azure Biosystems), images were captured using Licor Fc imaging system and the signal intensities were calculated using ImageStudio software.
    ImageStudio
    suggested: None
    Corrected counts were normalized by mouse PolyA+ enriched RNA, which was mapped to mouse genome using STAR (17) not allowing mismatches.
    STAR
    suggested: (STAR, RRID:SCR_004463)

    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

    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.

  3. Version published to 10.1101/2021.08.18.456855v1 on bioRxiv