Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

  1. Eric S. Pringle
  2. Brett A. Duguay
  3. Maxwell P. Bui-Marinos
  4. Rory P. Mulloy
  5. Shelby L. Landreth
  6. Krishna Swaroop Desireddy
  7. Stacia M. Dolliver
  8. Shan Ying
  9. Taylor Caddell
  10. Trinity H. Tooley
  11. Patrick D. Slaine
  12. Stephen L. Bearne
  13. Darryl Falzarano
  14. Jennifer A. Corcoran
  15. Denys A. Khaperskyy
  16. Craig McCormick

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Abstract

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting accumulation of full-length viral genomes, subgenomic RNAs and structural proteins. In ectopic expression models, we observed that 6-TG increased the electrophoretic mobility of Spike from diverse betacoronaviruses, matching the effects of enzymatic removal of N-linked oligosaccharides from Spike in vitro . SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on production of lentiviruses pseudotyped with SARS-CoV-2 Spike, yielding pseudoviruses deficient in Spike and unable to infect ACE2-expressing cells. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. Using biochemical and genetic approaches we demonstrated that 6-TG is a pro-drug that must be converted to the nucleotide form by hypoxanthine phosphoribosyltransferase 1 (HPRT1) to achieve antiviral activity. This nucleotide form has been shown to inhibit small GTPases Rac1, RhoA, and CDC42; however, we observed that selective chemical inhibitors of these GTPases had no effect on Spike processing or accumulation. By contrast, the broad GTPase agonist ML099 countered the effects of 6-TG, suggesting that the antiviral activity of 6-TG requires the targeting of an unknown GTPase. Overall, these findings suggest that small GTPases are promising targets for host-targeted antivirals.

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  1. Version published to 10.1371/journal.ppat.1010832
  2. Version published to 10.1101/2022.03.10.483772v3 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2022.03.10.483772: (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
    tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-BiP (Cell signaling Technologies (CST), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), anti-SARS-CoV-2 E (abbexa, abx226552), anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730)
    anti-puromycin ( Millipore-Sigma , MABE343
    suggested: None
    anti-OC43 N ( CoV
    suggested: None
    anti-BiP
    suggested: None
    anti-XBP1s
    suggested: None
    anti-CHOP
    suggested: (Cell Signaling Technology Cat# 2895, RRID:AB_2089254)
    anti-α-Tubulin ( CST
    suggested: None
    anti-SARS-CoV-2
    suggested: None
    anti-SARS-CoV-2 E
    suggested: None
    anti-SARS-CoV-2 M
    suggested: None
    anti-SARS-CoV-2 N
    suggested: None
    NBP3-05730
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HCT-8 cells were cultured as above with additional 1X MEM Non-Essential Amino Acids (NEAA, Gibco
    HCT-8
    suggested: None
    Baby hamster kidney (BHK-21) were maintained as above yet supplemented with 5% FBS.
    BHK-21
    suggested: ATCC Cat# CRL-6281, RRID:CVCL_1914)
    African green monkey kidney Vero’76 and human lung adenocarcinoma Calu-3 cells were maintained in DMEM (Sigma-Aldrich, D5796; St. Louis, MO, USA) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, 16000-044) and 1X Penicillin-Streptomycin (Gibco, 15140148).
    Calu-3
    suggested: None
    1A) or Vero E6 cells (SARS-CoV-2/SB3-TYAGNC, used in Figs.
    Vero E6
    suggested: None
    Stocks were titered by plaque assay on Vero E6 cells (68) or TCID50 on Vero’76 cells calculated using the Spearman-Kärber method.
    Vero’76
    suggested: None
    Stocks of human coronavirus OC43 (hCoV-OC43; ATCC, VR-1558) were propagated in Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    After 1 h, the inoculum was removed, replaced with DMEM/Pen/Strep/Gln containing 1% FBS and NEAA (HCT-8), 2% FBS (Calu-3), 2.5% FBS (Huh-7.5, 293A or 293T), or 5% FBS (BJ) supplemented with DMSO or drug at the indicated concentration, and maintained for an additional 23 h at the indicated temperature.
    Huh-7.5
    suggested: RRID:CVCL_7927)
    293A
    suggested: None
    pLJM1-ACE2-BSD was cloned by from cDNA of generated from Caco-2 cells.
    Caco-2
    suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
    SARS-CoV-2 Spike-pseudotyped lentiviruses: To generate Spike-pseudotyped lentivirus particles, 293T cells were seeded in a 10 cm dish and co-transfected with 3 µg pLJM1-Luc2, 2 µg pSPAX2 (a kind gift from Didier Trono; Addgene #12260), and 2 µg of pcDNA3-SΔ19 with PEI, then treated with 6-TG or DMSO as described above.
    293T
    suggested: None
    Recombinant DNA
    SentencesResources
    To generate 293A-ACE2 cells, 293A cells were stably transduced with a lentivirus vector encoding ACE2 (pLJM1-ACE2-BSD) or an empty vector control (pLJM1-B*) (67), then selected and maintained in 10 µg/mL Blasticidin S HCl (ThermoFisher, A11139030)
    pLJM1-ACE2-BSD
    suggested: None
    The pcDNA3-SARS-CoV-2-Spike plasmid contains a codon-optimized ORF for Spike from GenBank NC_045512 that was synthesized by GenScript (a kind gift from David Kelvin) then cloned between the KpnI and BamHI sites of pcDNA3.1(+).
    pcDNA3-SARS-CoV-2-Spike
    suggested: None
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    pcDNA3-Membrane (M) and pcDNA3-Envelope (E) were cloned from pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro and pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro (kind gifts from Neven Krogan, available from Addgene #141386 and #141385) using PCR to remove the 2xStrep epitope tag.
    pcDNA3-Membrane
    suggested: None
    pcDNA3-Envelope
    suggested: None
    pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro
    suggested: RRID:Addgene_141386)
    pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro
    suggested: RRID:Addgene_141385)
    pCMV3-MERS-FLAG was purchased from SinoBiologicals (VG40069-CF).
    pCMV3-MERS-FLAG
    suggested: None
    pLJM1-Luc2 used in generating Spike-pseudotyped lentiviruses was generated by cloning Luc2 from pGL4.26 (Promega) into pLJM1-B* (67).
    pGL4.26
    suggested: RRID:Addgene_68742)
    pLJM1-B*
    suggested: None
    Production of SARS-CoV-2 virus-like particles: To generate SARS-CoV-2 virus-like particles, 293T cells were seeded in a 10 cm dish and co-transfected with 2 µg of each pcDNA-Spike, pcDNA-M, pcDNA-E, and pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro (a kind gift from Neven Krogan, available from Addgene #141391) with PEI, then treated with 6-TG or DMSO as described above.
    pcDNA-Spike
    suggested: None
    pcDNA-M
    suggested: None
    pcDNA-E
    suggested: None
    pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro
    suggested: RRID:Addgene_141391)
    SARS-CoV-2 Spike-pseudotyped lentiviruses: To generate Spike-pseudotyped lentivirus particles, 293T cells were seeded in a 10 cm dish and co-transfected with 3 µg pLJM1-Luc2, 2 µg pSPAX2 (a kind gift from Didier Trono; Addgene #12260), and 2 µg of pcDNA3-SΔ19 with PEI, then treated with 6-TG or DMSO as described above.
    pSPAX2
    suggested: RRID:Addgene_12260)
    pcDNA3-SΔ19
    suggested: None
    Quantitation of SARS-CoV-2 Spike surface expression: 293T cells were co-transfected with pEGFP-N1 and plasmids expressing SARS-CoV-2 Spike (FL) or SARS-CoV-2 Spike-Δ19 for 24 h using FugeneHD (Promega).
    pEGFP-N1
    suggested: RRID:Addgene_172284)
    Gaussia luciferase secretion assays: For short-term treatments, 293T were transfected with pCMV-Gaussia Luc Vector (ThermoFisher Scientific, 16147) as described above.
    pCMV-Gaussia
    suggested: None
    For long-term treatments, 293T cells were co-transfected with pCMV-Gaussia Luc and pLJM1-Luc2 and treated 6 h post transfection.
    pLJM1-Luc2
    suggested: None
    Software and Algorithms
    SentencesResources
    Values were imported into GraphPad PRISM (v.9) and CC50 values were calculated by fitting a non-linear curve to the data.
    GraphPad PRISM
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Z-stacks were imaged on a Zeiss LSM880 and processed into maximum intensity projections using Zen Black (Zeiss).
    Zen
    suggested: None
    tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-BiP (Cell signaling Technologies (CST), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), anti-SARS-CoV-2 E (abbexa, abx226552), anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730)
    Millipore-Sigma
    suggested: None
    Graphics: Molecular models were created with NCBI PubChem (http://pubchem.ncbi.nlm.nih.gov/) The model presented in Fig.
    PubChem
    suggested: (PubChem, RRID:SCR_004284)
    http://pubchem.ncbi.nlm.nih.gov/
    suggested: (PubChem BioAssay, RRID:SCR_010734)
    7 was adapted from “Coronavirus Replication Cycle”, by BioRender.com (2022).
    BioRender
    suggested: (Biorender, RRID:SCR_018361)

    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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  4. Version published to 10.1101/2022.03.10.483772v2 on bioRxiv
  5. Version published to 10.1101/2022.03.10.483772v1 on bioRxiv