Fig 1.
Stress kinase activators and phosphorylation of eIF2α.
The chemical inducers used in this study (ArsNa, BTdCPU, DTT, and BEPP) and its target cellular kinase (HRI, PERK, and PKR) leading to eIF2α phosphorylation are indicated.
Fig 2.
USUV infection inhibits ArsNa-induced eIF2α phosphorylation in Vero cells.
(A) Vero cells were mock or USUV infected (MOI of 1), treated with ArsNa (0.5 mM), BTdCPU (20 μM), DTT (1 mM), or BEPP (10 μM) for 4 hours at 24 and 48 h p.i., and analyzed by immunofluorescence. Phosphorylated eIF2α was detected with anti-p-eIF2α antibody (green) and USUV-infected cells with anti-dsRNA antibody (red). Nuclei were stained with To-Pro-3 (blue). (B) Quantification of p-eIF2α fluorescence intensity in cells treated as in (A). Scale bars, 25 μm. Statistically significant differences were considered when P<0.05 and marked with an asterisk.(C) Vero cells were mock or USUV infected at an MOI of 1, and treated with ArsNa (0.5 mM) or the with same amount of DMSO for 4 hours at 48 h p.i. Cells were lysed and HRI was detected by western blot using an specific antibody. Membrane was reincubated with an anti-β-actin antibody as a control for protein loading.
Fig 3.
USUV infection inhibits ArsNa-induced eIF2α phosphorylation in Neuro2a cells.
(A) Neuro2a cells were mock or USUV infected (MOI of 1), treated with ArsNa (0.5 mM) or BTdCPU (20 μM), for 4 hours at 48 h p.i., and analyzed by immunofluorescence. Phosphorylated eIF2α was detected with anti-p-eIF2α antibody (green) and USUV-infected cells with anti-dsRNA antibody (red). Nuclei were stained with To-Pro-3 (blue). (B) Quantification of p-eIF2α fluorescence intensity in cells treated as in A. Scale bars, 25 μm. Statistically significant differences were considered when P<0.05 and marked with an asterisk.
Fig 4.
(A) Vero cells were mock-infected or infected with USUV (MOI of 1) and then treated with drug vehicle (DMSO), ArsNa (0.5 mM) or BTdCPU (20μM) for 4 hours at 48 h p.i. CellROX green Reagent was added at a final concentration of 5 μM and incubated for 30 minutes at 37°C, and cells were analyzed by immunofluorescence. Green fluorescent signal showed the ROS-mediated oxidation of the reagent. Nuclei were stained with To-Pro-3 (blue). Scale bars, 25 μm. (B) Quantification of CellROX fluorescent puncta in cells treated as in A. Data are means of three independent experiments. Statistically significant differences were considered when P<0.05 and marked with an asterisk. (C and D) Antioxidant activity of USUV infection. (C) Vero and (D) Neuro2a cells were mock-infected or infected with USUV at an MOI of 10 and left untreated. The conversion of DCFH-DA into DCF upon addition of a free radical initiator was measured by recording the fluorescence in a plate reader every 5 min. Antioxidant capacity was inversely proportional to fluorescence intensity. Quercetin (25 μM) was used as control of antioxidant activity. See methods for details.
Fig 5.
USUV infection inhibits ArsNa-induced SGs formation.
(A) Vero cells were mock- or USUV- infected at an MOI of 1, treated with ArsNa (0.5 mM), BTdCPU (20μM), DTT (1mM) or BEPP (10 μM) for 4 hours at 24 and 48 h p.i., and analyzed by immunofluorescence. SGs were detected with anti-G3BP1 antibody (green) and USUV-infected cells with anti-dsRNA antibody (red). Nuclei were stained with To-Pro-3 (blue). (B) Quantification of SG-positive cells in cells treated as in A. Scale bars, 25 μm. Statistically significant differences were considered when P<0.05 and marked with an asterisk.
Fig 6.
Effect of HRI activators on USUV multiplication.
(A) Cell viability was determined as ATP content in mock-infected cells treated with the drugs at 24 and 48 h. The dotted line indicates 80% of the viability of control cells; (B) Cells were infected with an MOI of 1 PFU/cell, and 24 or 48 h p.i. were treated with ArsNa (0.5 mM), or BTdCPU (20 μM) for 4 hours. Then, virus yields in culture supernatants were determined by plaque assay. Data represent the average of three independent experiments.