Figure 1.
DDX24 is a FADD-associating interferon inducible helicase.
(A) Schematic of human DDX24 (hDDX24) indicating helicase domains. (B) Schematic of human DDX24 promoter region, indicating STAT1 and IRF7 binding site. (C) Yeast two hybrid assay confirming FADD and DDX24 interactions. (D)293T cells were transiently transfected with c-Myc-DDX24 and FLAG–FADD or control plasmid. Lysates were immunoprecipitated (IP) and immunoblotted (IB) using antibodies to c-Myc or FLAG. (E) On the left, Immunoblot analysis of DDX24 in HUVEC cells treated with DDX24 siRNA or control ns (non-specific) siRNA. On the right, endogenous human DDX24 associates with FADD in HUVEC. Lysates of HUVEC cells were immunoprecipitated with anti-FADD or mouse IgG serum. The immunoprecipitates were analyzed by immunoblot with anti-DDX24 or anti-FADD (top). The expression levels of the endogenous DDX24 and FADD were detected by immunoblot analysis (bottom). (F) Schematic of human DDX24 (hDDX24) indicating helicase domains. 293T cells were transiently transfected with c-Myc-DDX24-FL, c-Myc-DDX24-N or control plasmid with FLAG-FADD, Lysates were immunoprecipitated (IP) using antibodies to FLAG and immunoblotted (IB) using antibodies to c-Myc. (G) Immunoblot analysis of DDX24 in varies human cells or cell lines and MEFs, normalized byGAPDH. (H) MEFs were left untreated or treated with mIFNβat 100 U/ml. Mouse ddx24 and rig-i mRNA were analyzed by RT-PCR. (I) MEFs were left untreated or treated with poly I:C for the indicated time. Mouse ddx24 mRNA was analyzed by RT-PCR. (J) MEFs were left untreated or treated with poly I:C, mIFNβ for the indicated time. Mouse DDX24 and β-actin were detected by immunoblot analysis. (K) HUVEC cells were left untreated or treated with hIFNβ at increasing does. Human DDX24 and β-actin were detected by immunoblot analysis. Data from (H)(I) are presented as means±s.e. from three independent experiments. * indicates P<0.05. ** indicates P<0.01.
Figure 2.
DDX24 inhibits dsRNA induced RLR signaling.
(A) DDX24 inhibits poly I:C-induced activation of the IFNβ promoter in a dose-dependent manner in MEFs. MEF cells transfected with vector or c-Myc-DDX24 (200 µg or 400 µg) were transfected with 2 µg/ml poly I:C overnight before testing for luciferase expression. Expression of c-Myc tagged DDX24 in MEFs was monitored using anti c-Myc antibody, normalized by β-actin. (B) DDX24 inhibits VSVdM-induced activation of the IFNβ promoter in a dose-dependent manner in MEFs. MEF cells transfected with vector or c-Myc-DDX24 (200 µg or 400 µg) were infected by VSVdM at MOI 10 overnight before testing for luciferase expression. (C) DDX24 inhibits poly I:C-induced endogenous ifnb transcription in MEFs. MEF cells transfected with vector or c-Myc-DDX24 (200 µg or 400 µg) were transfected with 2 µg/ml poly I:C for 6 hours before testing ifnb RNA by RT-PCR. (D) DDX24 inhibits VSVdM-induced endogenous ifnb transcription in MEFs. MEF cells transfected with vector or c-Myc-DDX24 (200 µg or 400 µg) were infected by VSVdM at MOI 10 for 6 hours before testing ifnb RNA by RT-PCR. (E) DDX24 inhibits poly I:C-induced endogenous IFNβ protein expression inMEFs. MEFs were transfected with control plasmid or DDX24 expressing plasmid. Twenty four hours after transfection, cells were transfected with 2 µg/ml poly I:C or left untreated overnight. Endogenous IFNβ were analyzed by ELISA. (F) DDX24 inhibits VSVdM-induced endogenous IFNβ protein expression inMEFs. MEFs were transfected with control plasmid or DDX24 expressing plasmid. Twenty four hours after transfection, cells were infected with VSVdM at MOI = 1 or left uninfected overnight. Endogenous IFNβ were analyzed by ELISA. Data are presented as means±s.e. from three independent experiments. * indicates P<0.05. ** indicates P<0.01.
Figure 3.
SiRNA-mediated knockdown of DDX24 enhances dsRNA induced RLR signaling.
(A) Effects of DDX24 RNAi on poly I:C-induced activation of the IFNβ promoter in 293T cells. 293T cells were transfected with ns or human ddx24 siRNA. Forty eight hours after transfection, cells were left untreated or transfected with poly I:C overnight before luciferase assays were performed. 293T cell lysates were analyzed by immunoblotting with the indicated antibodies to ensure the knockdown of hDDX24. (B) Effects of DDX24 RNAi on VSVdM-induced activation of the IFNβ promoter in 293T cells. 293T cells were transfected with ns or human ddx24 siRNA. Forty eight hours after transfection, cells were left untreated or infected with VSVdM overnight before luciferase assays were performed. (C) Effects of DDX24 RNAi on 6 hours treatment of poly I:C-induced endogenous ifnb transcription in MEFs by RT-PCR. MEF cell lysates were analyzed by immunoblotting with the indicated antibodies to ensure the knockdown of mDDX24. (D) Effects of DDX24 RNAi on 6 hours infection of VSVdM-induced endogenous ifnb transcription in MEFs by RT-PCR. (E) Effects of DDX24 RNAi on overnight treatment of poly I:C-induced endogenous IFNβ production in MEFs by ELISA. (F) Effects of DDX24 RNAi on overnight infection of VSVdM-induced endogenous IFNβ production in MEFs by ELISA. (G) Gene array indicating most up-regulated genes in MEF treated with poly I:C at 3 hours and 9 hours VS non treatment. Data from (A)(B)(C)(D)(E)(F) are presented as means±s.e. from three independent experiments. * indicates P<0.05. ** indicates P<0.01.
Figure 4.
SiRNA-mediated knockdown of DDX24 inhibits VSV replication.
(A) Loss of mDDX24 affects VSV-luc replication in MEFs. MEFs transfected with ns, mDDX24 or RIG-I siRNA for 72 hours were infected by VSV-luc at M.O.I. = 0.1 or 1. Eight hours and 24 hours post infection, luciferase activities from infected MEF cell lysate were detected. (B) Virus titer from supernatant of (A). (C) Loss of mDDX24 affects VSV-GFP replication in MEFs. Fluorescence microscopy (GFP) of ns, mDDX24 or mRIG-I siRNA treated MEFs following with VSV-GFP infection 24 hours post infection at M.O.I 1. (D) Knockdown efficiency check in MEF. DDX24 and RIG-I antibody are used to detect endogenous DDX24 or RIG-I. (E) Virus titer from supernatant of (C). (F) Loss of hDDX24 affects VSV replication in HUVECs. HUVECs transfected with ns or hDDX24 siRNA for 72 hours were infected by VSV-luc at M.O.I. = 0.1 or 1. Eight hours and 24 hours post infection, plaque assays were performed using supernatants from infected MEFs. HUVEC cell lysates were analyzed by immunoblotting with the indicated antibodies to ensure the knockdown of hDDX24. Data from (A)(B)(E)(F) are presented as means±s.e. from three independent experiments. * indicates P<0.05. ** indicates P<0.01.
Figure 5.
DDX24 can sequester RLR activator RNA.
(A) Schematic of human RIG-I and DDX24. (B) DDX24's binding specificity. 293T cells were transfected with c-Myc-tagged DDX24 for 24 hours before lysed. Pull-down assays were performed by incubating 293T lysate with various biotin-conjugated polynucleotides, and then precipitated with streptavidin beads. Bound proteins were analyzed by immunoblotting with anti-c-Myc antibody. (C) ssRNA transcribed from VSV-G cDNA were conjugated with biotin and utilized in pull-down assays similar to (B). (D) Endogenous DDX24 binds to VSV-G RNA. Pull-down assays were performed by incubating HUVEC cells lysate with biotion-VSV-G. Endogenous DDX24 were analyzed by immunoblotting with anti-DDX24 antibody. (E) DDX24 attenuates RIG-I's RNA binding activity. 293T cells were transfected with c-Myc-tagged DDX24 or FLAG-RIG-I as indicated for 24 hours before lysed. Pull-down assay were performed using biotin-VSV-G and bound proteins were applied to immunoblotting. (F) In vitro translated DDX24 binds to VSV-G RNA. Pull-down assays were performed by incubating in vitro translated DDX24 with biotion-VSV-G or non-labeled VSV-G. DDX24 were analyzed by immunoblotting with anti-c-Myc antibody. (G) In vitro translated DDX24 helicase C domain binds to VSV-G RNA. Pull-down assays were performed by incubating in vitro translated c-Myc-DDX24-N or c-Myc-DDX24-C with biotion-VSV-G or non-labeled VSV-G. DDX24mutants were analyzed by immunoblotting with anti-c-Myc antibody. (H) DDX24 inhibits dRIG-I-, dMDA5, IPS-1 and TBK-1 mediated IFNβ promoter activation. (I)(J) DDX24 blocks synergistical effect of FADD/RIP1 with RIG-I. 293T cells were transfected with reporter plasmid and variant plasmids as indicated. Activations of IFNβ promoter were detected 36 hours post transfection. (K) DDX24 does not affect p53 activation. 293T cells were transfected with reporter plasmid and variant plasmids as indicated. Activations of p53 promoter were detected 36 hours post transfection. Data from (H)(I)(J)(K) are presented as means±s.e. from three independent experiments. * indicates P<0.05. ** indicates P<0.01.
Figure 6.
DDX24 interacts with RIP1 and disrupts RLR's activation of IFN-dependent transcription factor IRF7.
(A) DDX24's effect on IRF3 phosphorylation. 293T cells transfected with vector or c-Myc-DDX24 were treated with 2 µg/ml poly I:C or infected by VSVdM at MOI 10 for 3 hours. Cell lysates were then prepared, and the dimerization and phosphorylation of IRF3 was analyzed by native or SDS PAGE. (B) DDX24's effect on IRF7 phosphorylation. Immunoblot for detecting IRF7 phosphorylation using 293T cells transfected with c-Myc-tagged DDX24, FLAG-tagged IRF7, TBK-1 and IKKi as indicated. (C) DDX24 interacts with RIP1. 293T cells were transfected with either c-Myc-tagged RIP1 or FLAG-DDX24 as indicated. After 24 h, cells were harvested, and lysates were subjected to co-IP and immunoblotting (IB) with the indicated antibodies. (D) DDX24's effect on RIG-I dependent IFNα4 promoter activation. 293T cells were transfected with IFNα4-luc reporter plasmid and variant plasmids as indicated. Activations of IFNα4 promoter were detected 36 hours post transfection. (E)(F) Loss of DDX24's effect on RIG-I dependent IFNα4 promoter activation. 293T cells were transfected with ns or DDX24 siRNA. Forty eight hours post transfection, cells were transfected with IFNα4-luc reporter plasmid and variant plasmids as indicated. Activations of IFNα4 promoter were detected 36 hours post transfection. (G)(H) DDX24 disrupts RIP1-IRF7 interaction. 293T cells were transiently transfected with variant plasmids as indicated and proper control plasmids. Thirty six hours post transfection, cell lysates were immunoprecipitated (IP) and immunoblotted (IB) using antibodies to c-Myc, FLAG or FADD. (I) Loss of DDX24 enhances RIP1-IRF7 interaction. 293T cells were transiently transfected with ns siRNA or DDX24 siRNA. Forty eight hours later, cells were transfected with variant plasmids as indicated and proper control plasmids. Twenty four hours post transfection, cell lysates were immunoprecipitated (IP) and immunoblotted (IB) using antibodies to c-Myc, FLAG or DDX24.Data from (D)(E)(F) are presented as means±s.e. from three independent experiments. * indicates P<0.05. ** indicates P<0.01.
Table 1.
Genotypes of embryos/mice derived from DDX24+/− intercrosses.