A prenylated dsRNA sensor protects against severe COVID-19

  1. Arthur Wickenhagen
  2. Elena Sugrue
  3. Spyros Lytras
  4. Srikeerthana Kuchi
  5. Marko Noerenberg
  6. Matthew L. Turnbull
  7. Colin Loney
  8. Vanessa Herder
  9. Jay Allan
  10. Innes Jarmson
  11. Natalia Cameron-Ruiz
  12. Margus Varjak
  13. Rute M. Pinto
  14. Jeffrey Y. Lee
  15. Louisa Iselin
  16. Natasha Palmalux
  17. Douglas G. Stewart
  18. Simon Swingler
  19. Edward J. D. Greenwood
  20. Thomas W. M. Crozier
  21. Quan Gu
  22. Emma L. Davies
  23. Sara Clohisey
  24. Bo Wang
  25. Fabio Trindade Maranhão Costa
  26. Monique Freire Santana
  27. Luiz Carlos de Lima Ferreira
  28. Lee Murphy
  29. Angie Fawkes
  30. Alison Meynert
  31. Graeme Grimes
  32. ISARIC4C Investigators
  33. Joao Luiz Da Silva Filho
  34. Matthias Marti
  35. Joseph Hughes
  36. Richard J. Stanton
  37. Eddie C. Y. Wang
  38. Antonia Ho
  39. Ilan Davis
  40. Ruth F. Jarrett
  41. Alfredo Castello
  42. David L. Robertson
  43. Malcolm G. Semple
  44. Peter J. M. Openshaw
  45. Massimo Palmarini
  46. Paul J. Lehner
  47. J. Kenneth Baillie
  48. Suzannah J. Rihn
  49. Sam J. Wilson

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Abstract

The heterogeneity of COVID-19 makes it challenging to predict the course of infection in an individual. Upon virus infection, interferons (IFNs) generate the initial signals for cellular defenses. Knowing that defects in IFN signaling are associated with more severe COVID-19, Wickenhagen et al . used IFN-stimulated gene expression screening on human lung cells from which they identified a gene for 2′-5′-oligoadenylate synthetase 1 (OAS1) (see the Perspective by Schoggins). OAS1 stimulates RNase L to inhibit the virus with a surprising degree of specificity, targeting the membranous organelles in which it replicates. In most mammals, OAS1 is attached to membranes by a prenyl group. However, billions of humans do not have the prenylated OAS1 haplotype, including many experiencing severe COVID-19. The same is true for horseshoe bats, prolific sources of betacoronaviruses, because of an ancient retrotransposition event. —CA

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  1. Version published to 10.1126/science.abj3624
  2. Rapid Reviews Infectious Diseases

    Roy Parker, James Burke

    Review 3: "A Prenylated dsRNA Sensor Protects Against Severe COVID-19 and is Absent in Horseshoe Bats"

    This preprint colleagues perform a screen to identify interferon-stimulated genes that inhibit SARS-CoV-2 replication. The authors deem the study design as reliable and recommended only minor revisions.

  3. Rapid Reviews Infectious Diseases

    Alexei Korennykh

    Review 2: "A Prenylated dsRNA Sensor Protects Against Severe COVID-19 and is Absent in Horseshoe Bats

    This preprint colleagues perform a screen to identify interferon-stimulated genes that inhibit SARS-CoV-2 replication. The authors deem the study design as reliable and recommended only minor revisions.

  4. Rapid Reviews Infectious Diseases

    Margo Brinton

    Review 1: "A Prenylated dsRNA Sensor Protects Against Severe COVID-19 and is Absent in Horseshoe Bats"

    This preprint colleagues perform a screen to identify interferon-stimulated genes that inhibit SARS-CoV-2 replication. The authors deem the study design as reliable and recommended only minor revisions.

  5. Rapid Reviews Infectious Diseases

    Strength of evidence

    Reviewers: Margo Brinton (Georgia State University) | 📗📗📗📗◻️
    Alexei Korennykh (Princeton) | 📗📗📗📗◻️
    Roy Parker (University of Colorado) | 📘📘📘📘📘
    James Burke (University of Colorado) | 📘📘📘📘📘

  6. ScreenIT

    SciScore for 10.1101/2021.05.05.21256681: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: This work (ethics approval number 32077020.6.0000.0005) was approved on May 2020 by the National Committee in Ethics and Research, Brazil. in COMISSÃO NACIONAL DE ÉTICA EM PESQUISA.
    IRB: This work (ethics approval number 32077020.6.0000.0005) was approved on May 2020 by the National Committee in Ethics and Research, Brazil. in COMISSÃO NACIONAL DE ÉTICA EM PESQUISA.
    Sex as a biological variableAs control tissue, FFPE lung of a healthy, 62-years old, male donor was used (NBP2-30182, Novusbio, cat. No 0028000B).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    IAV foci immunostaining was achieved using mouse the anti-influenza A virus nucleoprotein monoclonal antibody clone AA5H (BioRad, MCA400) and visualised with goat anti-mouse IgG (H+L)-HRP conjugate (BioRad, 1721011) and TrueBlue Peroxidase Substrate (KPL, 5510-0030) following standard plaque assay protocols as described previously (Turnbull et al., 2016).
    anti-influenza
    suggested: None
    MCA400
    suggested: (Bio-Rad Cat# MCA400, RRID:AB_2151884)
    anti-mouse IgG
    suggested: (Bio-Rad Cat# 172-1011, RRID:AB_11125936)
    Blots were probed with either antibodies raised against actin (mouse JLA20 hybridoma; courtesy of the Developmental Studies Hybridoma Bank, University of Iowa), OAS1 (rabbit polyclonal 14955-1-AP, Proteintech), OAS3 (rabbit polyclonal 21915-1-AP, Proteintech), or the rabbit anti-RNase L monoclonal antibody (Cell Signalling Technology, 27281).
    antibodies raised against actin
    suggested: None
    OAS1
    suggested: (Proteintech Cat# 14955-1-AP, RRID:AB_2158292)
    OAS3
    suggested: (Proteintech Cat# 21915-1-AP, RRID:AB_2876880)
    anti-RNase L
    suggested: None
    Thereafter, membranes were probed with species IgG-specific fluorescently labelled secondary antibodies goat anti-rabbit IgG or goat anti-mouse IgG (Thermo Scientific) and scanned using a LiCor Odyssey scanner.
    anti-rabbit IgG
    suggested: None
    Immunostaining was performed using a rabbit anti-OAS1 monoclonal antibody [clone D1W3A] (Cell Signaling Technology, 14498) and sheep anti-SARS-CoV-2-nsp5 antiserum, (https://mrcppu-covid.bio described in (Rihn et al., 2021)).
    anti-SARS-CoV-2-nsp5
    suggested: None
    Secondary antibody staining was performed with Alexa Fluor™ 488 Goat anti-Rabbit IgG and Alexa Fluor™ 594 Donkey anti Sheep IgG both at 1:1000 (Invitrogen).
    anti Sheep IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK-293T cells were propagated from lab stocks and A549-NPro cells were a kind gift of Prof. Richard E. Randall.
    HEK-293T
    suggested: None
    A549-NPro
    suggested: None
    Calu-3 cells were maintained in MEM supplemented with 10% FCS, 2 mM glutamine, 2 mM sodium pyruvate and 100 μM non-essential amino acids.
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    Indiana vesiculovirus (VSV) was a kind gift of Dr. Megan Stanifer and was propagated on BHK cells at low MOI as described previously (Rihn et al., 2019).
    BHK
    suggested: None
    Lentiviral vectors were produced by transfecting 293T cells as described previously (Rihn et al., 2019) and 0.45 μm-pore-size-filtered supernatant was used to transduce AAT cells and were selected using 2 μg/ml puromycin.
    293T
    suggested: None
    A549-ISRE::GFP cells (gift from Prof. Richard E.
    A549-ISRE::GFP
    suggested: None
    Virus inputs were normalized using plaque assays on VeroE6 cells to 500 PFU per well.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Recombinant DNA
    SentencesResources
    Retroviral vectors and cell modification: The lentiviral vector pSCRPSY (KT368137.1) has been previously described (Kane et al., 2016).
    pSCRPSY
    suggested: None
    pLV-EF1a-IRES-Puro (Addgene plasmid # 85132) was modified by PCR amplifying the tagRFP ORF (using pSCRPSY as template) flanked by directional SfiI sites which were further flanked by BamHI and EcoRI restriction sites (forward oligo: 5’-CTC TCG GAT CCG GCC GAG AGG GCC ATG AGC GAG CTG ATT AAG-3’ and reverse oligo: 5’-CTC TCG AAT TCG GCC AGA GAG GCC TCA CTT GTG CCC CAG-3’) and the BamHI-EcoRI fragment was subcloned into the vector to create the modified pLV-EF1a-IRES-Puro-SfiI-tagRFP construct.
    pLV-EF1a-IRES-Puro
    suggested: RRID:Addgene_85132)
    pLV-EF1a-IRES-Puro-SfiI-tagRFP
    suggested: None
    The cDNA corresponding to the ORFs of the following OAS genes (GenBank accession number): OAS1p46 (NM_016816), human OAS3 (NM_006187), macaque OAS1 (EF467665), bovine OAS1X (NM_178108), bovine OAS1Y (NM_001040606), bovine OAS1Z (AY650038), and Rhinolophus ferrumequinum OAS1 (XM_033097132 / Ensembl: ENSRFET00010016745) were chemically synthesised as gene-blocks with flanking SfiI sites (IDT DNA) and the SfiI fragment was subcloned into the modified pLV-EF1a-IRES-Puro-SfiI plasmid.
    pLV-EF1a-IRES-Puro-SfiI
    suggested: None
    To generate the human OAS1p42 sequence (in accordance with GenBank accession NM_002534), OAS1p46-c397a, and OAS1p42-CTIL sequences, the pLV-SfiI-OAS1p46 lentiviral vector plasmid was modified by overlap extension PCR (using primer pair 5’-CTC TCT GGC CGA GAG GGC CAT GAT GGA TCT CAG AAA TAC CCC AG-3’ and 5’-TCT CTC GGC CAG AGA GGC CTC AAG CTT CAT GGA GAG GGG CAG GGA TGA ATG GCA GGG AGG AAG CAG GAG GTC TCA CCA GCA GAA TCC AGG AGC TCA CTG GG-3’ for OAS1p42, primer pair 5’-CTC TCT GGC CGA GAG GGC CAT GAT GGA TCT CAG AAA TAC CCC AG-3’ and 5’-TCT CTC GGC CAG AGA GGC CTC AGA GGA TGG TGG CGG TCC AGT CCT CTT CTG CCT GTG GG-3’ for p46-c395a, and primer pair 5’-CTC TCT GGC CGA GAG GGC CAT GAT GGA TCT CAG AAA TAC CCC AG -3’ and 5’-TCT CTC GGC CAG AGA GGC CTC AGA GGA TGG TGC AAG CTT CAT GGA GAG GGG CAG GGA TGA ATG GCA GGG AGG AAG CAG GAG GTC TCA CCA GCA GAA TCC AGG AGC TC ACT GGG -3’ for p42-CTIL) and the SfiI-fragment was subcloned in place of OAS1p46 in the pLV lentiviral vector plasmid described above.
    pLV-SfiI-OAS1p46
    suggested: None
    pLV lentiviral
    suggested: None
    Software and Algorithms
    SentencesResources
    CRISPR guides were designed using the CHOPCHOP online tool (https://chopchop.cbu.uib.no).
    https://chopchop.cbu.uib.no
    suggested: (CHOPCHOP, RRID:SCR_015723)
    Post-acquisition, the contrast of images within each set were optimised using Zen software (Carl Zeiss) to equal degrees for the vector, p42, p42-CTIL and p46 C397A samples, whereas the histogram maximum was increased independently in the p46 sample shown in (Figure 4) to prevent oversaturation in the green channel due to strong perinuclear concentration.
    Zen
    suggested: None
    DIGS uses a nucleotide or amino acid sequence probe to perform a BLAST similarity search through genome assemblies.
    DIGS
    suggested: None
    We first used the nucleotide sequence of the syntenic region of R. ferrumequinum to human exon 7 (Ensembl) and the adjacent 580bp region with the detected LTR insertion until homology resumes to the human genome as a probe (Supplementary digs_probes.fas).
    Ensembl
    suggested: (Ensembl, RRID:SCR_002344)
    Matches were aligned using MAFFT v7.453 (Katoh and Standley, 2013) and inspected for covering all regions of the probe (Supplementary Table 1).
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    PDE analysis: To examine the diversity of PDE proteins encoded by coronaviruses we first constructed an HMMER protein profile.
    HMMER
    suggested: (Hmmer, RRID:SCR_005305)
    The alignment was then manually curated using Bioedit based on the homology described in the literature.
    Bioedit
    suggested: (BioEdit, RRID:SCR_007361)
    The EMBOSS getorf program (Rice et al., 2000) was used to extract the translated sequences of all methionine starting open reading frames (ORFs) with length above 100 nucleotides from the filtered virus genome dataset.
    EMBOSS
    suggested: (EMBOSS, RRID:SCR_008493)
    The study was registered at https://www.isrctn.com/ISRCTN66726260. Pre-processed and STAR (Dobin et al., 2013) mapped paired-end reads of 499 whole-blood patient transcriptomes with known disease outcomes from the ISARIC4C study were analysed to stratify mild (hospitalised but not ICU-admitted patients) and severe (ICU admitted and/or deceased) patients further into p46 +ve and p46 -ve groups.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Analyses were implemented using IBM SPSS Statistics version 25 (IBM Corp. Armonk, USA).
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    ISRCTN66726260NANA

    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.

  7. Version published to 10.1101/2021.05.05.21256681v1 on medRxiv