Because DHX29 contains two ATP binding motifs (Walker A and B) in the DEXD motif-containing domain , we next sought to determine whether these ATP binding motifs are necessary for DHX29 to sense HMW Poly(I:C) by introducing mutations in the Walker A and B sites of DHX29 (WAM-DHX29 and WBM-DHX29). Unlike full-length DHX29, WAM-DHX29 and WBM-DHX29 failed to bind to HMW Poly(I:C) or to increase the HMW Poly(I:C)-binding ability of MDA5 (Fig 6F). Consistent with these observations, we showed that like DHX29a and DHX29b, WAM-DHX29 and WBM-DHX29 did not increase but rather inhibited MDA5-mediated ISRE-Luc activity, as compared to full-length DHX29 (Fig 6G). The inhibitory effects of WAM-DHX29, WBM-DHX29, DHX29a, and DHX29b on HMW Poly(I:C)-induced ISRE-Luc activity were increased with increasing amounts of gene expression (S6D Fig), thus exerting a dominant negative effect on DHX29 function as an RNA sensor. Taken together, these results indicate that DHX29 functions as a sensor to recognize HMW Poly(I:C) and to interact with MDA5. The N-terminal domain of DHX29 is required for MDA5 binding, whereas the DEXD (Walker A and B)-containing domain is required for RNA binding. Thus, both the N-terminal and DEXD/helicase domains of DHX29 are required for its function as an RNA co-sensor to promote MDA5-mediated type I IFN signaling.