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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 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.
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ppat-1003721-g002: 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.

Mentions: To evaluate the importance of DDX24 in possible innate immune signaling regulation, we overexpressed DDX24 in MEF cells and confirmed that the type I IFN inducer, poly I:C, could initiate the transcription of an IFNβ promoter driven luciferase construct (Figure 2A). However, we noted that the overexpression of DDX24 could inhibit poly I:C's ability to activate the IFNβ promoter (Figure 2A). Similarly, DDX24 was seen to inhibit the ability of vesicular stomatitis virus (VSVdM, with a defective matrix protein that enables virus-mediated type I IFN production) to activate IFNβ-luciferase production [34] (Figure 2B). This was confirmed at the level of IFNβ mRNA expression and endogenous protein expression (Figure 2C–2F). Thus, DDX24 may negatively regulate innate immune signaling processes that activate the type I IFN promoter.


DDX24 negatively regulates cytosolic RNA-mediated innate immune signaling.

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814876&req=5

ppat-1003721-g002: 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.
Mentions: To evaluate the importance of DDX24 in possible innate immune signaling regulation, we overexpressed DDX24 in MEF cells and confirmed that the type I IFN inducer, poly I:C, could initiate the transcription of an IFNβ promoter driven luciferase construct (Figure 2A). However, we noted that the overexpression of DDX24 could inhibit poly I:C's ability to activate the IFNβ promoter (Figure 2A). Similarly, DDX24 was seen to inhibit the ability of vesicular stomatitis virus (VSVdM, with a defective matrix protein that enables virus-mediated type I IFN production) to activate IFNβ-luciferase production [34] (Figure 2B). This was confirmed at the level of IFNβ mRNA expression and endogenous protein expression (Figure 2C–2F). Thus, DDX24 may negatively regulate innate immune signaling processes that activate the type I IFN promoter.

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