FIG. 4.

Activation of the transcription factor IRF-3 in oligodendrocytes by MHV infection. N20.1 cells were infected with MHV-A59/GFP at an MOI of 10 (Virus), mock infected as a negative control (Mock), or transfected with poly(I:C) as a positive control [I:C or Poly(I:C)]. (A and B) IRF-3 is phosphorylated by MHV infection. At 2 and 4 h p.i. or 4 h posttransfection, cell lysates were isolated for detection of phosphorylated and total IRF-3 (P-IRF-3 and T-IRF-3, respectively) by Western blot analysis. β-Actin was used as an internal control. Lanes M, mock infection. (C) IRF-3 is translocated to the nucleus during MHV infection. At 4 h p.i. or posttransfection, cells were fixed and the intracellular localization of IRF-3 was visualized by IFA staining with an antibody to IRF-3. (D and E) Activation of IRF-3 and NF-κB by MHV infection. At 2, 4, and 8 h p.i. or posttransfection, nuclear extracts were prepared and the IRF-3 or NF-κB activity was determined with the respective ELISA-based assay, in which the oligonucleotide containing the IRF-3 or NF-κB consensus binding site is immobilized on the microplate for capturing the activated transcription factor. The amount of the bound complexes was determined with a microplate reader as optical density at 450 nm (OD₄₅₀). The values represent the means of the readings from three samples, and error bars are the standard deviations of the means. The asterisks indicate statistical significance (P < 0.05) between the comparison groups.