FIG 2.

rsT3D induces higher levels of IRF3 activation than rsT1L in SVECs. (A) SVECs were mock infected (M) or infected with rsT1L or rsT3D at an MOI of 1, 10, or 100 PFU/cell. Whole-cell lysates were prepared at the indicated times, and immunoblot analysis was performed to detect phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, or reovirus proteins. (B) SVECs were mock infected or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell, and cytoplasmic and nuclear fraction lysates were prepared at 6 h. Immunoblot analysis was performed to detect phosphorylated IRF3 (p-IRF3), total IRF3, β-actin, α-tubulin, lamin A/C, or reovirus proteins. (C) SVECs were transfected with an IRF3 firefly luciferase reporter (p55C1BLuc) and a Renilla luciferase control. At 24 h, cells were mock infected, transfected with poly I·C (2 μg), or infected with rsT1L or rsT3D at an MOI of 100 PFU/cell. Luciferase expression was measured at 24 h posttransfection. Results are presented as the mean ratio of firefly luciferase activity to Renilla luciferase activity for triplicate samples from three independent experiments ± SD. **, P < 0.01; ***, P < 0.001 (as determined by one-way ANOVA test). (D) SVECs were infected with rsT1L or rsT3D at an MOI of 100 PFU/cell. At 24 h, cells were fixed and stained for reovirus antigen (green). Nuclei were stained with DAPI (blue). (E) Quantification of the percentage of infected cells in panel D. Results are presented as the mean number of infected cells from triplicate samples from two independent experiments. (F) SVECs were infected with rsT1L or rsT3D at an MOI of 100 or 1,000 PFU/cell. At 6 h, whole-cell lysates were prepared and immunoblot analysis was performed to detect phosphorylated IRF3 (p-IRF3), β-actin, or reovirus proteins.