FIG 8.

The smallpox virus antiviral IBT induces antiviral granules. (A) HeLa and U2OS cells were infected with WT-VACV at an MOI of 10 in the presence of 0.05% DMSO, 1 μg/ml 1-β-d-arabinofuranosylcytosine (AraC), 50 μM isatin β-thiosemicarbazone (IBT), 50 μg/ml rifampin (Rifamp), or 5 μM ST-246 and fixed at 7 hpi. Coverslips were stained with antibodies to G3BP1 (red), hybridized with F9L mRNA probes (green) and DAPI (blue) as described in the legend of Fig. 5. (A) Example field of view for cells treated with IBT, showing the localization of G3BP1 and F9L mRNA to AVGs at cytoplasmic viral factories. (B) Cells were grown as described above for panel A, with the addition of 10 μg/ml cycloheximide during the final hour of growth (6 to 7 hpi). Quantification of infected cells showing factory-associated G3BP1/CAPRIN1 granules is shown in the merged panel (91%; n > 50 from each of 2 biological replicates). (C) Chart showing quantification of infected cells with AVGs after treatment with DMSO (0.0%), AraC (1.4% ± 1.4%), IBT (94.5% ± 2.2%), rifampin (6.8% ± 3.8%), and ST-246 (3.4% ± 1.0%). Only IBT treatment showed a significant incidence of AVG formation compared to treatment with DMSO alone (****, P < 0.0001). All experiments included >50 cells from at least 2 biological replicates. (D) HeLa cells were treated with 0.05% DMSO, 500 μM arsenite, 50 μM arsenite, or 50 μM IBT for 24 h before the MTT assay was performed. Absorbance values normalized to DMSO values (relative light units [RLU]) showed a statistically significant increase in cell metabolism for treatment with 50 μM IBT (85.0% ± 3.7%) compared to treatment with either 500 μM arsenite (30.2% ± 1.0%; P = 0.0004) or 50 μM arsenite (50.7% ± 4.5%; P = 0.013). (E and F) Viral growth was determined after the addition of 50 μM IBT by a plaque assay (log₁₀ PFU/ml) (E) and a fluorescence reporter assay (0.17% ± 1.4% RFU; P < 0.0001) (F).