Abstract
Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3 X-linked (DDX3X) is an ATP-dependent RNA helicase, In addition to involvement of eukaryotic gene expression regulation, mammalian DDX3X has recently been found to regulate IFN-β production via the adaptor MAVS mediated cascade signaling. In our studies, we demonstrated that chicken DDX3X (chDDX3X) is also involved in the IFN-β regulation, and demonstrated that chDDX3X regulated IFN-β via an essential adaptor chicken stimulator of IFN genes (chSTING). We found that chDDX3X overexpression in DF-1 cells induced expression of IFN-β and inhibited avian influenza virus (AIV) or Newcastle disease virus (NDV) replication. Knockdown of chDDX3X decreased the production of IFN-β induced by RNA analog polyinosinic-polycytidylic acid and increased viral yield. Furthermore, chDDX3X was identified as a potential chSTING-interacting protein by co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). And exogenous Co-IP in transfected cells with or without virus-stimulations further confirmed the interaction between chDDX3X and chSTING. With the gene overexpression and RNA interference studies, the chDDX3X was confirmed to be located upstream of chSTING and activate IFN-β via the chSTING-chTBK1-chIRF7-IFN-β signaling axis. In brief, our results suggest that chDDX3X is an important IFN-β mediator and is involved in RNA- and RNA virus-mediated chDDX3X-chSTING-IFN-β signaling pathway.
Highlights
Innate immunity acts as the first line of defense against invading organisms
We found that the anti-Flag mAb-immunoprecipitated protein complexes were recognized by chicken DDX3X (chDDX3X) mAb and the interaction between chicken stimulator of IFN genes (chSTING) and chDDX3X was strengthened with viral stimulation in DF-1 cells (Figure 1E).These results confirmed that chDDX3X interacts with chSTING
Through Co-IP and LC-MS/MS, and Co-IP with or without virus stimulations experiments in DF-1 cells, we identified chDDX3X as a chSTING-interacting protein (Figures 1A–E)
Summary
Innate immunity acts as the first line of defense against invading organisms. Innate immune responses are activated by host pattern-recognition receptors (PRRs), which recognize conserved molecules presenting in certain pathogen classes, collectively referred to pathogen-associated molecular patterns (PAMPs) [1,2,3]. The main PRR groups that recognize viral nucleic acids are Toll-like receptors (TLRs), retinoic acid inducible gene I (RIG-I)-like receptors, which are part of the DExD/H family of RNA helicases, and cytoplasmic DNA receptors [4]. DDX3X, as well as the well-known RNA recognition sensor RIG-I ( known as DDX58) and MDA5, belongs to the DEAD-box helicase family. This family has at least eight conserved motifs
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