Several recent studies show that MDA5 forms filaments upon binding to dsRNA, which is critical for the activation of the IFN-β signaling pathway [47, 48]. To determine whether DHX29 promotes the aggregation of MDA5 filaments along dsRNA, we performed electrophoretic mobility shift assays (EMSAs). Although DHX29 and MDA5 protein alone could bind to dsRNA oligonucleotides, dsRNA-binding activity in both mature (higher band) and intermediate forms (lower band) was enhanced when DHX29 and MDA5 were combined (Fig 7A). To further confirm the binding enhancement, we utilized atomic force microscopy (AFM) to image the RNA-protein complex. DHX29 was able to bind to Poly(I:C) but did not form filaments (Fig 7B). However, DHX29 promoted the aggregation of MDA5 filaments on Poly(I:C) dsRNA (Fig 7B). Because the mica plate flattens at the molecular level, the height (vertical distance) attribution could be used to represent the diameter of the RING structure of the protein-RNA complex (Fig 7C). We obtained 10 individual “section mode” analyses of the RNA-protein complexes. Compared with the 2 nm diameter of dsRNA alone (standard height for double strand HMW Poly[I:C]), the diameter of the dsRNA-MDA5-DHX29 complex was around 4 nm, and the diameter of both the dsRNA-MDA5 and dsRNA-DHX29 complexes was around 3 nm (Fig 7C). These results suggest that DHX29 interacts with dsRNA and MDA5 to form the aggregation of DHX29-MDA5 along dsRNA, by which DHX29 promotes MDA5 activation.