Abstract
The human DEAD-box protein 3 (DDX3) has been reported as a positive regulator and functions in the induction of type I interferon signaling. We elucidated the function of DDX3 in the positive regulation of IFNB production in non-pDC cells. We found that DDX3 regulates virus-induced activation of IFNB at the level of IRF-3. However, it does not affect conventional innate signaling, including IRF-3 phosphorylation, dimerization, or nuclear translocation of IRF-3, but has some downstream events after IRF-3 phosphorylation. Co-immunoprecipitation analyses revealed that DDX3 interacts with IRF-3 through its DNA-binding domain and promotes IRF-3-mediated IFNB promoter activation. DDX3 does not affect the formation of the IRF-3/p300/CBP complex. Instead, ChIP and EMSA assay revealed that DDX3 promotes the recruitment of IRF-3 and transcriptional co-activator p300/CBP to the IFNB promoter. The ATP binding pocket of DDX3 is involved in this association and is essential for the transcriptional activation. Taken together, our study demonstrates that DDX3 plays an important role in guiding a transcription factor complex formed by antiviral signaling to the target gene promoter.
Highlights
Activation of IRF-3 involves a cascade of events: phosphorylation at Ser 386, homodimer formation, nuclear translocation, and assembly of a complex containing p300/CBP on the regulatory elements of type I IFN promoter to turn on transcription[7–12]
To further substantiate the biological role of DDX3 in the innate antiviral response, we generated DDX3-deficient 293T and HeLa cells by CRISPR/Cas[9] gene editing. Both DDX3-deficient 293T and HeLa cells contain non-functional N-terminally truncated proteins (Supplementary Fig. 1a). These cells were transfected with 5′-triphosphate double-stranded RNA (5′ppp-dsRNA), a ligand for retinoic acid-inducible gene I (RIG-I) or high molecular weight (HMW) poly (I:C), a ligand for MDA5 or infected with Sendai virus (SeV) or Newcastle disease virus (NDV)
Our study demonstrated that all the steps leading to the activation of IFNB gene were comparable in the presence or absence of DDX3 (Figs. 3, 5b), except that the IRF motif-DNA binding activity of the transcriptional factor complex composed of IRF-3 dimer and p300/CBP, was undetectable in the absence of DDX3 (Fig. 5a)
Summary
Innate immunity is the first line of defense against invading pathogens. Host cells recognize the pathogenassociated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), including Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and intracellular DNA sensors, and initiate antiviral r esponses[1]. Upon sensing cytoplasmic viral RNAs, N-terminal caspase activation and recruitment domain (CARD) of RIG-I and MDA5 interact with mitochondrial signaling adapter (MAVS), which subsequently recruit the downstream proteins kinases TBK1 or IKKε, which phosphorylate and activate IRF-36. IRF-3 is the most important factor in the regulation of virus-induced interferon (IFN) gene activation. IRF-3 is activated to promote antiviral responses. There have been several reports claiming that DDX3 is involved in virus-induced signaling leading to IFN production. Virus immune evading factor K7 and interacts with IKKε to facilitate IRF-3 activation[24]. Another report identified DDX3 as an interacting protein of TBK1 and like IKKε, TBK1 phosphorylates D DX326. DDX3 facilitates this pathway to activate IRF-727; the biological functions of DDX3 in innate antiviral immunity are not fully understood
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