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DDX24 negatively regulates cytosolic RNA-mediated innate immune signaling.

Ma Z, Moore R, Xu X, Barber GN - PLoS Pathog. (2013)

Bottom Line: Here, we report that a type I IFN inducible DExD/H helicase, DDX24, exerts a negative-regulatory effect on RLR function.These events preferentially impeded IRF7 activity, an essential transcription factor for type I IFN production.Our data provide a new function for DDX24 and help explain innate immune gene regulation, mechanisms that may additionally provide insight into the causes of inflammatory disease.

View Article: PubMed Central - PubMed

Affiliation: Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

ABSTRACT
RIG-I-Like Receptors (RLRs) sense cytosolic viral RNA to transiently activate type I IFN production. Here, we report that a type I IFN inducible DExD/H helicase, DDX24, exerts a negative-regulatory effect on RLR function. Expression of DDX24 specifically suppressed RLR activity, while DDX24 loss, which caused embryonic lethality, augmented cytosolic RNA-mediated innate signaling and facilitated RNA virus replication. DDX24 preferentially bound to RNA rather than DNA species and influenced signaling by associating with adaptor proteins FADD and RIP1. These events preferentially impeded IRF7 activity, an essential transcription factor for type I IFN production. Our data provide a new function for DDX24 and help explain innate immune gene regulation, mechanisms that may additionally provide insight into the causes of inflammatory disease.

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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.
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ppat-1003721-g006: 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.

Mentions: The regulation of IRF3 and NF-κB pathways are complex though are known to involve RIG-I/MDA5 invoking TBK1 to principally phosphorylate IRF3 which then along with activation of the NF-κB pathway activates IFNβ transcription [3]. These events produce type I IFN-inducible IRF7 that binds to and activates several IFNα genes to augment type I IFN production in a positive feedback manner [35]. To additionally evaluate the mechanisms of DDX24 activity, we inquired whether DDX24 affected IRF3 or IRF7 activity. However, we observed that DDX24 did not affect poly I:C or VSVdM dependent IRF3 phosphorylation or dimerization (Figure 6A). Furthermore, we did not observe an inhibition of constitutively activated IRF3(SA)–mediated induction of IFNβ-luc during overexpression of DDX24 (Figure S5B and Figure S5C). To next evaluate the influence of DDX24 on IRF7 function, we examined the effects of DDX24 on an IFNα4 promoter driving luciferase which is strongly activated by IRF7 rather than IRF3 [36]. This experiment indicated that overexpression of DDX24 in 293T cells inhibited the ability of RIG-I and IRF7 to fully activate the IFNα4 promoter (Figure 6D). Conversely, loss of DDX24 in 293T cells by RNAi treatment enhanced RIG-I and IRF7's ability to activate the IFNα4 promoter (Figure 6E). Overexpression of FADD was observed to facilitate IRF7 signaling, which was significantly increased in the absence of DDX24 (Figure 6F). To extend this analysis, we evaluated whether DDX24 could affect TBK1/IKKi's ability to phosphorylate IRF7. Co-expression analysis confirmed that DDX24 could affect IRF7 phosphorylation (Figure 6B). Thus, DDX24 could influence IRF7 function, which is a pivotal positive regulator of type I IFN production.


DDX24 negatively regulates cytosolic RNA-mediated innate immune signaling.

Ma Z, Moore R, Xu X, Barber GN - PLoS Pathog. (2013)

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.
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ppat-1003721-g006: 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.
Mentions: The regulation of IRF3 and NF-κB pathways are complex though are known to involve RIG-I/MDA5 invoking TBK1 to principally phosphorylate IRF3 which then along with activation of the NF-κB pathway activates IFNβ transcription [3]. These events produce type I IFN-inducible IRF7 that binds to and activates several IFNα genes to augment type I IFN production in a positive feedback manner [35]. To additionally evaluate the mechanisms of DDX24 activity, we inquired whether DDX24 affected IRF3 or IRF7 activity. However, we observed that DDX24 did not affect poly I:C or VSVdM dependent IRF3 phosphorylation or dimerization (Figure 6A). Furthermore, we did not observe an inhibition of constitutively activated IRF3(SA)–mediated induction of IFNβ-luc during overexpression of DDX24 (Figure S5B and Figure S5C). To next evaluate the influence of DDX24 on IRF7 function, we examined the effects of DDX24 on an IFNα4 promoter driving luciferase which is strongly activated by IRF7 rather than IRF3 [36]. This experiment indicated that overexpression of DDX24 in 293T cells inhibited the ability of RIG-I and IRF7 to fully activate the IFNα4 promoter (Figure 6D). Conversely, loss of DDX24 in 293T cells by RNAi treatment enhanced RIG-I and IRF7's ability to activate the IFNα4 promoter (Figure 6E). Overexpression of FADD was observed to facilitate IRF7 signaling, which was significantly increased in the absence of DDX24 (Figure 6F). To extend this analysis, we evaluated whether DDX24 could affect TBK1/IKKi's ability to phosphorylate IRF7. Co-expression analysis confirmed that DDX24 could affect IRF7 phosphorylation (Figure 6B). Thus, DDX24 could influence IRF7 function, which is a pivotal positive regulator of type I IFN production.

Bottom Line: Here, we report that a type I IFN inducible DExD/H helicase, DDX24, exerts a negative-regulatory effect on RLR function.These events preferentially impeded IRF7 activity, an essential transcription factor for type I IFN production.Our data provide a new function for DDX24 and help explain innate immune gene regulation, mechanisms that may additionally provide insight into the causes of inflammatory disease.

View Article: PubMed Central - PubMed

Affiliation: Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.

ABSTRACT
RIG-I-Like Receptors (RLRs) sense cytosolic viral RNA to transiently activate type I IFN production. Here, we report that a type I IFN inducible DExD/H helicase, DDX24, exerts a negative-regulatory effect on RLR function. Expression of DDX24 specifically suppressed RLR activity, while DDX24 loss, which caused embryonic lethality, augmented cytosolic RNA-mediated innate signaling and facilitated RNA virus replication. DDX24 preferentially bound to RNA rather than DNA species and influenced signaling by associating with adaptor proteins FADD and RIP1. These events preferentially impeded IRF7 activity, an essential transcription factor for type I IFN production. Our data provide a new function for DDX24 and help explain innate immune gene regulation, mechanisms that may additionally provide insight into the causes of inflammatory disease.

Show MeSH
Related in: MedlinePlus