293T cells were co-transfected with expression vectors encoding proteins RIG-I, MDA5, MAVS and TBK1 together with increasing amounts of expression vector that encodes DHX29 protein along with the ISRE or NF-ĸB luciferase reporter. We found that DHX29 specifically enhanced activation of the luciferase reporters induced by MDA5, but not others (Fig 5B). To further determine whether the endogenous interaction between DHX29 and MDA5 occur under physiological conditions, we treated THP-1 or mDHX29-expressing MEFs with intracellular HMW Poly(I:C) or EMCV infection. Immunoprecipitation and immunoblot analysis revealed no interaction between endogenous DHX29 and MDA5 in THP-1 cells stimulated with intracellular LMW Poly(I:C) (S5F Fig). The endogenous DHX29-MDA5 interaction was only observed following intracellular HMW Poly(I:C) treatment or EMCV infection (Fig 5C–5E). Consistently, DHX29 was detected in a complex with MDA5 in the presence of HMW Poly(I:C) by in vitro pull-down experiments using purified DHX29 and MDA5 proteins (Fig 5F). Colocalization of DHX29 and MDA5 after HMW Poly(I:C) or EMCV stimulation was detected by confocal imaging (Fig 5G). Specifically, the interaction of DHX29 with MDA5 was observed starting from 150 min after HMW Poly(I:C) stimulation analyzed by time-recording fluorescent microscopy (Fig 5H).