Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions
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.