Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions
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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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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
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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), … 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
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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 , MABE343suggested: Noneanti-OC43 N ( CoVsuggested: Noneanti-BiPsuggested: Noneanti-XBP1ssuggested: Noneanti-CHOPsuggested: (Cell Signaling Technology Cat# 2895, RRID:AB_2089254)anti-α-Tubulin ( CSTsuggested: Noneanti-SARS-CoV-2suggested: Noneanti-SARS-CoV-2 Esuggested: Noneanti-SARS-CoV-2 Msuggested: Noneanti-SARS-CoV-2 Nsuggested: NoneNBP3-05730suggested: NoneExperimental Models: Cell Lines Sentences Resources HCT-8 cells were cultured as above with additional 1X MEM Non-Essential Amino Acids (NEAA, Gibco HCT-8suggested: NoneBaby hamster kidney (BHK-21) were maintained as above yet supplemented with 5% FBS. BHK-21suggested: 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-3suggested: None1A) or Vero E6 cells (SARS-CoV-2/SB3-TYAGNC, used in Figs. Vero E6suggested: NoneStocks were titered by plaque assay on Vero E6 cells (68) or TCID50 on Vero’76 cells calculated using the Spearman-Kärber method. Vero’76suggested: NoneStocks of human coronavirus OC43 (hCoV-OC43; ATCC, VR-1558) were propagated in Vero cells. Verosuggested: 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-7.5suggested: RRID:CVCL_7927)293Asuggested: NonepLJM1-ACE2-BSD was cloned by from cDNA of generated from Caco-2 cells. Caco-2suggested: 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. 293Tsuggested: NoneRecombinant DNA Sentences Resources 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-BSDsuggested: NoneThe 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-Spikesuggested: NonepcDNA3.1suggested: 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-Membranesuggested: NonepcDNA3-Envelopesuggested: NonepLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Purosuggested: RRID:Addgene_141386)pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Purosuggested: RRID:Addgene_141385)pCMV3-MERS-FLAG was purchased from SinoBiologicals (VG40069-CF). pCMV3-MERS-FLAGsuggested: NonepLJM1-Luc2 used in generating Spike-pseudotyped lentiviruses was generated by cloning Luc2 from pGL4.26 (Promega) into pLJM1-B* (67). pGL4.26suggested: RRID:Addgene_68742)pLJM1-B*suggested: NoneProduction 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-Spikesuggested: NonepcDNA-Msuggested: NonepcDNA-Esuggested: NonepLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Purosuggested: 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. pSPAX2suggested: RRID:Addgene_12260)pcDNA3-SΔ19suggested: NoneQuantitation 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-N1suggested: 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-Gaussiasuggested: NoneFor long-term treatments, 293T cells were co-transfected with pCMV-Gaussia Luc and pLJM1-Luc2 and treated 6 h post transfection. pLJM1-Luc2suggested: NoneSoftware and Algorithms Sentences Resources Values were imported into GraphPad PRISM (v.9) and CC50 values were calculated by fitting a non-linear curve to the data. GraphPad PRISMsuggested: (GraphPad Prism, RRID:SCR_002798)Z-stacks were imaged on a Zeiss LSM880 and processed into maximum intensity projections using Zen Black (Zeiss). Zensuggested: Nonetween-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-Sigmasuggested: NoneGraphics: Molecular models were created with NCBI PubChem (http://pubchem.ncbi.nlm.nih.gov/) The model presented in Fig. PubChemsuggested: (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). BioRendersuggested: (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.
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