FIG 2.

Antiviral granule formation is enhanced during alterations of translation initiation. HeLa and U2OS cells were infected with WT-VACV at an MOI of 10. At 6 hpi, cells were treated with PBS, 500 μM arsenite (Ars), 1 μM thapsigargin (Thap), 1 μM hippuristanol (Hipp), or 1 μM rohinitib (RHT) and fixed an hour later at 7 hpi. Alternatively, cells were pretreated with 200 U/ml beta 1 interferon for 24 h before infection. Cells were stained with antibodies to G3BP1, TIA1, or CAPRIN1 and with DAPI. (A) Schematic describing parallel methods to alter translation initiation with small molecules. The activation of multiple stress kinases results in the phosphorylation of the alpha subunit of eIF2, preventing the formation of the ternary complex necessary for translation initiation. Alternatively, eIF4A is inhibited by hippuristanol and RHT in an eIF2α-independent manner. (B) Whole-cell extracts from mock-infected and infected cells treated with 500 μM Ars were separated by SDS-PAGE and blotted with an antibody against phosphorylated eIF2α (S51) (i) and total eIF2α (ii). A band densitometry comparison of the phosphorylated form normalized to total eIF2α is shown between gel images. (C) Example images for G3BP1 (red) (middle) and DAPI (blue) (right) labeling and merged images (left) for PBS (i)-, arsenite (ii)-, thapsigargin (iii)-, hippuristanol (iv)-, rohinitib (v)-, and beta 1 interferon (vi)-treated cells. N, cell nuclei; arrows, factories with AVGs; solid arrowheads, factories without granules; open arrowheads, stress granules (bar = 5 μm). (D) Quantification of infected cells with AVGs after treatment with arsenite (91.3% ± 1.9% [mean ± SEM]), thapsigargin (32.6% ± 7.4%), hippuristanol (77.9% ± 9.6%), and RHT (92.8% ± 1.6%) but not IFN (4.1% ± 0.7%) (P = 0.9997) showed a highly significant (****, P < 0.0001) increase compared to cells treated with PBS alone (5.3% ± 0.8%). All experiments included >50 cells from at least 2 biological replicates. ns, not significant. (E) HeLa cells were infected with WT-VACV, and 500 μM arsenite was added 1 h before fixation at 4 to 8 hpi. (i) Cells were stained as described above for panel C (bar = 5 μm). (ii and iii) Magnified dashed boxes containing viral factories with AVGs are shown in orthogonal views as a maximum-intensity projection, including the top view (x and y, bar = 5 μm) (ii) and side view (z, bar = 2 μm) (ii). Borders of DAPI-stained factories are denoted with dashed lines.