Fig 1.
Thiopurines 6-thioguanine and 6-thioguanosine inhibit coronavirus replication.
(A) Structures of thiopurines used this study in comparison to guanine. (B) Calu-3 cells were infected with SARS-CoV-2 at an MOI of 0.1 then treated with 6-thioguanine (6-TG), 6-thioguanosine (6-TGo), or 6-mercaptopurine (6-MP). Supernatants were harvested after 48 h and stored at -80°C until titering on Vero’76 cells. Mock-infected cells were similarly treated with 6-TG, 6-TGo, 6-MP, or DMSO vehicle control for 48 h before testing cell viability with CellTiter 96 AQueous One (n = 3 ± SEM). Dotted line indicates Limit of Detection. (C) Summary table of 50% Cytoxic Concentration (CC50), 50% Effective Concentration (EC50), and Selectivity Index (SI) calculated for (A-C). (D) AlamarBlue cell viability assay of hTert-BJ, HCT-8, and Huh-7.5 cells treated with 6-TG (n = 3±SEM). (E-H) TCID50 assays for (E) HCoV-OC43 infected HCT-8 cells and (F) HCoV-229E infected Huh-7.5 cells. Cells were infected with an MOI of 0.1 then treated with tunicamycin (Tm), 6-TG, 6-TGo, 6-MP, or DMSO (n≥3 ± SEM, statistical significance was determined by one-way ANOVA). hTERT-BJ cells were infected with HCoV-OC43 (G) or HCoV-229E (H) at an MOI of 0.1 and treated with 6-TG, Tm, or DMSO. Supernatants were harvested after 23 h and stored at -80°C before titering on BHK-21 or Huh7.5 (n = 3–4 ± SEM, statistical significance was determined by one-way ANOVA). LOD = Limit of Detection for virus titer. (*, p<0.05; **, p<0.01; ns, non-significant).
Fig 2.
6-TG reduces CoV protein and mRNA accumulation.
(A-C) 293T cells were infected with HCoV-OC43 at an MOI of 0.1 then treated with 10 μM 6-TG or DMSO vehicle control. Supernatant and total RNA from the cells were harvested at the times indicated and stored at -80°C until (A) titering on BHK-21 or (B) quantification of viral genomes by RT-qPCR. Cell monolayers were harvested for (C) RT-qPCR analysis of viral RNA accumulation (n = 3 statistical significance was determined by paired ratio t-test; *, p<0.05; **, p<0.01; ns, non-significant). (D) 293T cells were infected as in (A) then treated with 5 or 10 μM 6-TG or DMSO. Lysates were harvested 24 h after infection and were probed by western blotting as indicated. (E-F) 293T cells were grown on coverslips then infected with HCoV-OC43 and treated with 6-TG as in (A), then fixed and immunostained for (E) N or (F) dsRNA. Nuclei are counterstained with Hoechst. Scale bar = 20 μm.
Fig 3.
6-TG inhibits Spike glycosylation and cleavage.
(A) Calu-3 cells were infected with SARS-CoV-2 at an MOI of 7, then treated with 6-TG or DMSO. Lysates were harvested 48 h after infection and were probed by western blotting as indicated. Concentrations in legend are in μM. (B) Huh-7.5 cells were infected with SARS-CoV-2 at an MOI of 5, then treated with 6-TG or DMSO. Lysates were harvested 48 h after infection and were probed by western blotting as indicated. Concentrations in legend are in μM. (C) 293T cells were transfected with plasmids encoding SARS-CoV-2 Spike (S), Membrane (M), Envelope (E), Nucleoprotein (N) with a C-terminal 2xStrep tag, or empty vector (EV) then treated with 10 μM 6-TG or DMSO vehicle control. Lysates were harvested 24 h after transfection and probed by western blotting as indicated. (D) 293T cells were transfected with SARS-CoV-2 Spike or EV then treated with 6-TG or DMSO vehicle control. Lysates were harvested 24 h after transfection and were probed by western blotting as indicated. Concentrations in legend are in μM. (E) AlamarBlue cell viability assay of 6-TG treated 293T cells (n = 3±SEM). (F) 293T cells were transfected with plasmids encoding SARS-CoV-2 Spike, a Spike mutant lacking the Furin cleavage site between S1 and S2 (S0), or EV then treated with 10 μM 6-TG or DMSO vehicle control. Lysates were harvested 24 h after transfection, treated with PNGase F to remove N-linked glycans, and probed by western blotting as indicated. (G) as in (C) except 293T cells were transfected with plasmids encoding C-terminal FLAG-tagged Spike (S-FL) or HCoV-MERS Spike (MERS). (H) as in (C) except 293T cells were transfected with plasmids encoding FLAG-tagged Spike from HCoV-OC43 (OC43) or HCoV-HKU1 (HKU1).
Fig 4.
6-TG reduces surface expression of Spike but does not globally limit secretion.
(A) AlamarBlue cell viability assay of 293T cells treated with brefeldin A (BFA) (n = 4±SEM). (B) 293T cells were transfected with plasmid encoding Gaussia luciferase for 18 h then treated with 6-TG, BFA or DMSO vehicle control for 6 h. Supernatants were recovered from the cells and measured for luciferase activity. (C) 293T cells were transfected with plasmids encoding firefly luciferase and Gaussia luciferase then treated with 6-TG, BFA, or DMSO. After 24 h, supernatants were removed and analyzed as in (B). Cell lysate was harvested in Reporter Lysis Buffer and stored at -80°C until luciferase and Gaussia luciferase activities were measured. (For B and C, n = 3 ± SEM, statistical significance was determined by paired t-test compared to DMSO-treated cells; *, p<0.05; **, p<0.01; ***, p<0.001; ns, non-significant). (D) 293T cells were transfected with plasmids encoding SARS-CoV-2 Spike or EV then treated with 6-TG, BFA, or DMSO. Lysates were harvested 24 h after transfection in 2x Laemmli buffer and were probed by western blotting as indicated. (E) 293T cells were co-transfected with plasmids encoding EGFP and either Spike (S), Spike with 19 residue C-terminal truncation (SΔ19), or EV and then treated with 10 μM 6-TG or DMSO. After 24 h, cells were harvested, surface-stained for Spike then fixed prior to analysis by flow cytometry. EGFP+ cells were gated for analysis of the number of Spike+ cells and Median Fluorescent Intensity (MFI) (n = 4±SEM statistical significance was determined by paired t-test between 6-TG and DMSO treated cells; *, p<0.05).
Fig 5.
6-TG stimulates an assembly defect and inhibits Spike virion incorporation.
(A) 293T cells were transfected with equal quantities of SARS-CoV-2 S, M, E, and N plasmids or empty vector (EV) then treated with 10 μM 6-TG or DMSO vehicle control. Lysates were harvested 48 h after transfection and probed by western blotting as indicated. (B) Virus-like particles from supernatants of cells transfected in (A) were concentrated by ultracentrifugation. Samples from two independent VLP preparations were probed by western blotting as indicated. (C) 293T cells were transfected with plasmids encoding S, E, M, and N in a 1:2:2:1 ratio, substituting one of the structural proteins for EV as indicated, treated with 6-TG or DMSO then processed as in (A). (D) SARS-CoV-2 Spike pseudotyped, luciferase-expressing lentivirus particles were concentrated by ultracentrifugation. Samples from three independent lentivirus preparations were probed by western blotting as indicated. (E) Genomes from three independent lentivirus preparations were quantified by RT-qPCR (n = 3, statistical significance was determined by paired t-test; ns, non-significant). (F) 293A cells stably expressing ACE2 or empty vector control were transduced with lentivirus from three independent preparations. After 24 h, lysates were harvested and measured for luciferase activity (n = 3, statistical significance was determined by two-way ANOVA; *, p<0.05; ns, non-significant). (G) 293A cells were infected HCoV-OC43 at an MOI of 0.1, then treated with 6-TG or DMSO. Supernatants were concentrated as in (D) before virions were fixed and imaged by TEM with negative staining. Five virions from both 6-TG- and DMSO-treated samples are shown at 150,000 X magnification. Scale bar = 100 nm. Arrowhead indicates examples of Spike proteins extending from virion.
Fig 6.
Conversion of 6-TG by HPRT1 is essential for antiviral activity and defects in Spike maturation.
(A) HPRT1 catalyses a reaction between 6-TG and phosphoribosyl diphosphate (PRPP) to generate 6-thioguanosine monophosphate (6-TGMP) and pyrophosphate products. 6-TG methylated at the N9 nitrogen (6-TG-Me) was designed to be resistant to processing by HPRT1. (B) 293T cells were infected with HCoV-OC43 at an MOI of 0.1 then treated with 6-TG or 6-TG-Me. Supernatants were harvested at 24 hpi and stored at -80°C until titering on BHK-21 cells (n = 3 statistical significance was determined by paired ratio t-test; *, p<0.05; **, p<0.01; ns, non-significant). (C) 293T cells were transfected with SARS-CoV-2 Spike vector or an empty vector control then treated with 6-TG, 6-TG-Me, or DMSO vehicle control. Lysates were harvested 24 h after transfection and probed by western blotting as indicated. (D) 293T cells (parental), non-targeting (NT) CRISPR control cells, or two independent CRISPR-edited HPRT1 knockout cell lines (HPRT1-KO1 and -KO2) were infected with HCoV-OC43 at an MOI of 0.1 for 1 h prior to treatment with DMSO or 10 μM 6-TG for the remaining 23 h. Lysates were prepared 24 h and analyzed by western blotting as indicated. (E) As in (D) but cell supernatants were harvested at 24 h and titered as in (B) (n = 6 ±SEM, statistical significance was determined by paired ratio t test; ***, p<0.001; ****, p<0.0001; ns, non-significant, LOD = Limit of Detection.). (F) The cell lines in (D) were transfected with plasmids encoding codon-optimized HCoV-OC43-Spike or an empty vector followed by treatment with DMSO or 10 μM 6-TG. Lysates were prepared at 24 h and analyzed by western blotting as indicated.
Fig 7.
6-TG inhibits Spike maturation through GTPase inhibition.
(A) AlamarBlue cell viability assay of 293T cells treated Rac1 Inhibitor V (Rac1iV), Rhosin, CASIN, or ML099 (n = 3±SEM). (B) Summary table of 50% cytotoxic concentration (CC50) and inhibitor target of compounds tested in (A). (C) 293T cells were transfected with SARS-CoV-2 Spike vector or an empty vector control (EV) then treated with 6-TG, GTPase inhibitors, or DMSO vehicle control. Concentrations in legend are in μM. Lysates were harvested 24 h after transfection and were probed by western blotting as indicated. (D) 293T cells were transfected with plasmids encoding SARS-CoV-2 Spike or EV control then co-treated with 6-TG, 100 μM ML099 GTPase agonist, or DMSO. Lysates were harvested 24 h after transfection and were probed by western blotting as indicated. Concentrations in legend are in μM. (E) as in (D) except some samples were pre-treated (Pre) for 4 h with ML099. (F) HCT-8 and 293A were infected with an MOI of ~0.1 then treated with 10 μM 6-TG and/or 100 μM ML099 or DMSO vehicle control. After 24 h, the supernatants were harvested and stored at -80°C until titering on BHK-21 (n = 3 ± SEM, statistical significance was determined by one-way ANOVA; *, p<0.05; **, p<0.01; ns, non-significant). LOD = Limit of Detection for virus titer.
Fig 8.
6-TG inhibits multiple essential steps in virus replication and assembly.
6-Thioguanine (6-TG) is converted into 6-thioguanosine monophosphate (6-TGMP) by HPRT1, which can be subsequently processed into diphosphate (6-TGDP) and triphosphate forms (6-TGTP) (collectively known as 6-TGNP). HPRT1-dependent conversion of 6-TG to a nucleotide form is required for antiviral activity, which comprises defects in accumulation of viral RNAs and proteins, as well as defects in Spike glycosylation and incorporation into viral particles. The model presented in Fig 8 was adapted from “Coronavirus Replication Cycle”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates.
Table 1.
Primer sequences for RT-qPCR analysis.
Table 2.
Primers used for cloning and PCR mutagenesis.