Additional experimental evidence document that the phosphorylation level of IRF3 induced by TBK1 is not affected by DHX29 in both WT and MAVS knockout cell (S5B Fig), All together the results obtained from luciferase reporter assay and interaction data in RIG-I knockout cell showed the similar pattern as observed in WT cell (Fig 5A and 5B, S5C and S5D Fig). Cumulatively, all these data suggest that DHX29 specifically functions in concert with MDA5 and acts upstream of MAVS. To determine whether the interaction between DHX29 and MDA5 is dependent upon ligand stimulation, we treated DHX29- and MDA5-expressing 293T cells with intracellular HMW Poly(I:C) and found that the interaction between DHX29 and MDA5 was induced following intracellular HMW Poly(I:C) treatment (S5E Fig). Furthermore, DHX29 KD decreased the MDA5-MAVS interaction in response to HMW Poly(I:C) stimulation (S5J Fig). Henceforth, our mechanistic studies bolster us to suggest that DHX29 specifically binds to MDA5 and promotes the MDA5-MAVS interaction to enhance type I IFN signaling. To identify the interacting domains between MDA5 and DHX29, we generated different deletion constructs of MDA5, and tested for their abilities to interact with DHX29 (Fig 5I). DHX29 interacted with full-length MDA5 and the CARD domain-containing N-terminal fragment (N-MDA5), but not with M-MDA5 or MC-MDA5 (Fig 5I).