Chickens are the natural host of Newcastle disease virus (NDV) and avian influenza virus (AIV). The discovery that the RIG-I gene, the primary RNA virus pattern recognition receptor (PRR) in mammals, is naturally absent in chickens has directed attention to studies of chicken RNA PRRs and their functions in antiviral immune responses. Here, we identified Asp-Glu-Ala-Asp (DEAD)-box helicase 1 (DDX1) as an essential RNA virus PRR in chickens and investigated its functions in anti-RNA viral infections. The chDDX1 gene was cloned, and cross-species sequence alignment and phylogenetic tree analyses revealed high conservation of DDX1 among vertebrates. A quantitative RT-PCR showed that chDDX1 mRNA are widely expressed in different tissues in healthy chickens. In addition, chDDX1 was significantly upregulated after infection with AIV, NDV, or GFP-expressing vesicular stomatitis virus (VSV-GFP). Overexpression of chDDX1 in DF-1 cells induced the expression of IFN-β, IFN-stimulated genes (ISGs), and proinflammatory cytokines; it also inhibited NDV and VSV replications. The knockdown of chDDX1 increased the viral yield of NDV and VSV and decreased the production of IFN-β, which was induced by RNA analog polyinosinic-polycytidylic acid (poly[I:C]), by AIV, and by NDV. We used a chicken IRF7 (chIRF7) knockout DF-1 cell line in a series of experiments to demonstrate that chDDX1 activates IFN signaling via the chIRF7 pathway. Finally, an in-vitro pulldown assay showed a strong and direct interaction between poly(I:C) and the chDDX1 protein, indicating that chDDX1 may act as an RNA PRR during IFN activation. In brief, our results suggest that chDDX1 is an important mediator of IFN-β and is involved in RNA- and RNA virus-mediated chDDX1-IRF7-IFN-β signaling pathways.

鸡是新城疫病毒 (NDV) 和禽流感病毒 (AIV) 的天然宿主。RIG-I 基因，哺乳动物的主要 RNA 病毒模式识别受体 (PRR)，在鸡中自然不存在，这一发现将注意力转向了对鸡 RNA PRR 及其在抗病毒免疫反应中的功能的研究。在这里，我们将 Asp-Glu-Ala-Asp (DEAD)-box 解旋酶 1 (DDX1) 鉴定为鸡中必不可少的 RNA 病毒 PRR，并研究了其在抗 RNA 病毒感染中的功能。chDDX1 基因被克隆，跨物种序列比对和系统发育树分析揭示了脊椎动物中 DDX1 的高度保守性。定量RT-PCR显示chDDX1 mRNA在健康鸡的不同组织中广泛表达。此外，chDDX1 在感染 AIV、NDV、或表达 GFP 的水泡性口炎病毒 (VSV-GFP)。DF-1 细胞中 chDDX1 的过表达诱导了 IFN-β、IFN 刺激基因 (ISG) 和促炎细胞因子的表达；它还抑制 NDV 和 VSV 复制。chDDX1 的敲低增加了 NDV 和 VSV 的病毒产量，并减少了 IFN-β 的产生，后者由 RNA 类似物聚肌苷酸 (poly[I:​​C])、AIV 和 NDV 诱导。我们在一系列实验中使用鸡 IRF7 (chIRF7) 敲除 DF-1 细胞系来证明 chDDX1 激活 IFN 信号 chDDX1 的敲低增加了 NDV 和 VSV 的病毒产量，并减少了 IFN-β 的产生，后者由 RNA 类似物聚肌苷酸 (poly[I:​​C])、AIV 和 NDV 诱导。我们在一系列实验中使用鸡 IRF7 (chIRF7) 敲除 DF-1 细胞系来证明 chDDX1 激活 IFN 信号 chDDX1 的敲低增加了 NDV 和 VSV 的病毒产量，并减少了 IFN-β 的产生，后者由 RNA 类似物聚肌苷酸 (poly[I:​​C])、AIV 和 NDV 诱导。我们在一系列实验中使用鸡 IRF7 (chIRF7) 敲除 DF-1 细胞系来证明 chDDX1 激活 IFN 信号通过chIRF7 通路。最后，一个体外pulldown 分析显示 poly(I:C) 和 chDDX1 蛋白之间存在强烈而直接的相互作用，表明 chDDX1 可能在 IFN 激活过程中充当 RNA PRR。简而言之，我们的结果表明 chDDX1 是 IFN-β 的重要介质，并参与 RNA 和 RNA 病毒介导的 chDDX1-IRF7-IFN-β 信号通路。