To understand the molecular mechanisms by which DHX29 enhances MDA5-mediated type I IFN signaling, we show that DHX29 has the ability to bind to HMW Poly(I:C) with a strong binding affinity compared with the low HMW Poly(I:C)-binding affinity of MDA5. This is in agreement with previous studies showing that MDA5 has a poor binding affinity for HMW Poly(I:C) and viral dsRNA [1, 15]. Coexpression of DHX29 and MDA5 markedly increases the binding capacity of MDA5 to HMW Poly(I:C), suggesting that DHX29 and MDA5 function as co-sensors for detecting EMCV infection. Further experiments demonstrate that the DEXD (containing Walker A and B ATP binding motifs) and helicase domains of DHX29 are responsible for HMW Poly(I:C) binding. Thus, it appears that DHX29 specifically interacts with MDA5 through its N-terminus and binds to HMW Poly(I:C) through its C-terminal DEXD and helicase domains, thus bringing MDA5 and HMW Poly(I:C) together. Indeed, our EMSA and AFM results show that the dsRNA-binding activity of DHX29 and MDA5 is much better than MDA5 alone. Overall, further analysis is needed to clarify whether the helicase activity of DHX29 is required for its function as co-sensor for MDA5-dsRNA. The potential role of DHX29 in the modulating of ATPase activity and oligomerization of MDA5 [57–59] requires further investigation, although our study strongly indicates that DHX29 bring HMW Poly(I:C) and MDA5 together for enhanced dsRNA binding capacity and consequent type I IFN signaling. Although DHX29 mainly functions as a core component of translation initiation complexes required for the translation of mRNA with 5´-structured UTR under normal physiological conditions, it may bring these structured RNAs in close proximity to MDA5 to promote MDA5-RNA binding and MDA5 aggregation along dsRNA after EMCV infection or HMW Poly(I:C) treatment, which in turn leads to the activation of MDA5 for consequent downstream type I IFN signaling.