FIG. 7.

Role of NF-κB in MHV-induced IFN-α/β production in oligodendrocytes. (A) Activation of NF-κB is partially mediated through RIG-I. N20.1 cells were transfected with the RIG-I siRNAs or nonspecific control siRNA (CsiRNA) or mock transfected. At 24 h posttransfection, cells were infected with MHV-A59/GFP at an MOI of 10 or mock infected. At 8 h p.i., nuclear extracts were isolated for determining the NF-κB activity with an ELISA-based assay kit as described in Materials and Methods. The asterisk indicates statistical significance (P < 0.05) between the comparison groups. (B) Optimization of NF-κB inhibitor. N20.1 cells were treated with NF-κB p65 decoy peptide inhibitor or the nonspecific control peptide inhibitor at 50 to 200 μM or mock treated for 1 h prior to infection. Cells were then infected with MHV-A59/GFP at an MOI of 10 or mock infected for 2 h in the presence of the inhibitors, and nuclear extracts were then prepared for determining the NF-κB activity as described for panel A. “Mock” indicates mock treatment and mock infection; “Virus” indicates virus infection only. (C and D) Effect of RIG-I knockdown on IFN-α/β induction. N20.1 cells were pretreated with NF-κB p65 inhibitor or control inhibitor at 150 μM and infected with the virus as described for panel B. At various time points as indicated, culture medium was harvested for determining IFN-α/β protein levels by ELISA. The results are expressed as the mean picograms per ml for three independent experiments. Error bars indicate standard deviations of the means.