DHX29 functions an RNA co-sensor and interacts with MDA5 for enhanced RNA recognition and type I IFN signaling.

<p>(<b>A</b>) Schematic of DHX29 truncated forms (<i>top panel</i>). 293T cells were transfected with <i>Flag-MDA5</i> and full-length <i>HA-DHX29</i> (<i>FL-DHX29</i>), <i>HA-DHX29a</i>, <i>HA-DHX29b</i>, <i>HA-DHX29c</i>, or <i>HA-DHX29d</i> plasmids. Immunoprecipitation with anti-HA beads and immunoblotting with anti-HA and anti-Flag antibodies were performed (<i>bottom panel</i>). <b>(B</b>) 293T cells were cotransfected with ISRE-luciferase (Luc) and <i>FL-DHX29</i>, empty vector (vec), <i>DHX29a</i>, <i>DHX29b</i>, <i>DHX29c</i>, or <i>DHX29d</i> plasmids and stimulated with IC HMW Poly(I:C). ISRE-Luc activity was normalized to the Renilla luciferase internal control and presented as the fold increase relative to stimulated FL-DHX29 control cells. (<b>C</b>) Cell lysates were prepared from 293T cells transfected with full-length <i>HA-DHX29 or HA-DHX29a or HA-DHX29c</i> alone or in combination with <i>Flag-MDA5</i>, and then incubated with biotin-labeled HMW Poly(I:C) for 4 h. Immunoprecipitation was performed with anti-biotin beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (<b>D</b>) Purified HA-DHX29 in the presence or absence of Flag-MDA5 was incubated with biotin-labeled LMW Poly(I:C) or HMW Poly(I:C) for 4 h. Pull-down experiments were performed with NeutrAvidin beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (<b>E</b>) Purified Flag-MDA5 in the presence or absence of BSA (control), FL-DHX29, DHX29a, DHX29b, DHX29c, or DHX29d protein was incubated with HMW Poly(I:C) for 4 h. Pull-down experiments were performed with anti-biotin beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (<b>F</b>) Schematic of DHX29 mutations in walker A (WAM-DHX29) and walker B (WBM-DHX29) sites. Purified Flag-MDA5 in the presence or absence of BSA (control), HA-DHX29, HA-WAM-DHX29, HA-WBM-DHX29 protein was incubated with HMW Poly(I:C) for 4 h. Immunoprecipitation with NeutrAvidin beads and immunoblotting with anti-Flag or anti-HA antibody were performed. <b>(G</b>) 293T cells co-transfected with ISRE-Luc and BSA (control), HA-DHX29, WAM-DHX29, or WBM-DHX29 were stimulated with IC HMW Poly(I:C). ISRE-Luc activity was normalized to the Renilla luciferase internal control and presented as the fold decrease relative to stimulated FL-DHX29 control cells. Data from (<b>B</b> and <b>G</b>) are representative of three independent experiments and plotted as the mean ± s.d. *<i>P</i> < 0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001 vs. the corresponding control. NS, not significant.