FIG. 2.

DEN and reoviruses trigger the innate immune response independently of RIG-I or MDA5. (A) wt, RIG-I^−/−, or MDA5^−/− mouse fibroblasts were either mock infected or infected with DEN2 at a multiplicity of infection of 1. (Left) Extracts derived from cells that were mock infected (M) or infected with DEN2 for the indicated times (in hours) were analyzed by immunoblotting for the abundance of IRF-3-responsive genes, RIG-I, MDA5, DEN2 NS1, and actin (used as a control). (Right) At 16 h postinfection, cells were fixed and stained with primary antibodies specific for IRF-3, followed by fluorescent-dye-conjugated secondary antibodies. Cellular distribution of IRF-3 (green) and 4′,6′-diamidino-2-phenylindole (DAPI)-stained nuclei (blue) was analyzed by immunofluorescence microscopy. (B) wt, RIG-I^−/−, or MDA5^−/− mouse fibroblasts were either mock infected (M) or infected with either reovirus T3D or reovirus T1L at a multiplicity of infection of 25. At the indicated times postinfection (in hours), cells were harvested and the extracts analyzed by immunoblotting. We note that a significantly longer exposure time was necessary for the detection of ISG54 and ISG56 in reovirus T1L-infected cells. (C) RIG-I^−/− mouse fibroblasts were infected with lentivirus particles expressing either a shRNA to MDA5 (shRNA 998, 2067, 2911, 3265, or 3547) or a nontargeting shRNA control (Neg). At 36 h postinfection, cells were either mock infected (−) or infected (+) with either DEN (left) or reovirus T3D (right) at multiplicities of infection of 1 and 25, respectively. Cells were harvested 48 h postinfection (84 h after infection with lentivirus) and the extracts analyzed by immunoblotting. For controls, extracts of RIG-I^−/− cells that were mock treated (M) or treated with exogenous IFN-β were also subjected to immunoblot analysis. **MDA5 indicates an image of MDA5 protein expression taken after a significantly longer exposure time.