To determine the specific role of DHX29 protein domains in the type I IFN pathway, four different DHX29 constructs (DHX29a [1–530 aa], DHX29b [1–830 aa], DHX29c [530–1369 aa], and DHX29d [830–1369 aa]) were generated and tested for their abilities to interact with MDA5 (Fig 6A). DHX29a and DHX29b, like full-length (FL) DHX29, were capable of binding to MDA5, whereas DHX29c and DHX29d failed to do so (Fig 6A), suggesting that the N-terminus (1–530 aa) of DHX29 is essential for DHX29 to interact with MDA5. In Fig 5I, DHX29 was shown to interact with N-MDA5. To confirm DHX29 interacts with MDA5 through N-DHX29 (DHX29a) and N-MDA5, we immunoprecipitated DHX29a with FL-MDA5 or deletions of MDA5, and found that DHX29a interacted only with FL- or N-MDA5 (S6A Fig). We next tested each of the four DHX29 constructs for their abilities to enhance type I IFN signaling using the ISRE-Luc reporter. The DHX29a and DHX29b constructs did not increase but rather inhibited intracellular HMW Poly(I:C)- or MDA5-induced ISRE-Luc activity, whereas DHX29c and DHX29d had no effect on the ISRE-luc activity compared with the vector control (Fig 6B and S6B Fig), suggesting that full-length DHX29 is required for its ability to enhance MDA5-mediated type I IFN signaling.