Characterizing DExD/H box helicases as viral sensors in human dendritic cells

描述（由申请人提供）：在过去十年中，使用基因组和遗传方法的主要努力已经鉴定了两大类用于感测微生物核酸的先天免疫受体：Toll样受体（TLR：TLR 3、7、8、9）和视黄酸诱导基因I样解旋酶（RLH：RIG-1、LGP 2、MDA-5）。然而，基因组学和遗传学方法在我们对这些受体如何结合核酸以及是否存在额外的受体或辅助受体的理解上留下了重大空白。我们决定打开这个“黑匣子”，采取生物化学的方法，直接分离和表征微生物核酸结合蛋白在人浆细胞样树突状细胞（pDC）的CpG-DNA下拉和质谱肽测序实验。我们鉴定了DExD/H-box解旋酶家族的两个新成员DHX 36和DHX 9，其分别特异性结合人pDC中的CpG-A和CpG-B。在pDC细胞系中通过siRNA敲低DHX 36表达与超过50%的1型IFN应答降低相关，并且消除了由CpG-A诱导的IRF 7核转位，并且敲低DHX 9表达与TNF和IL-6应答降低相关，并且阻断了由CpG-B诱导的NF-κ B p50核转位。DExD/H解旋酶家族有50多个成员。基于这些初步数据和其他病毒传感器，包括RIG-I样解旋酶，（RIG-1、LGP 2、MDA-5）和Dicer都属于DExD/H解旋酶家族，本研究将验证我们的中心假设：1）DExD/H-box解旋酶DHX 36和DHX 9可能代表pDC中新的TLR 9非依赖性DNA传感器;和2）DExD/H解旋酶家族（59个成员）可能在抗病毒先天免疫中发挥比以前认为的更广泛的作用。) 公共卫生相关性：通过CpG-DNA下拉和质谱肽测序，我们鉴定了两种新的DExD/H盒解旋酶家族成员DHX 36和DHX 9，其在人pDC中分别特异性结合并响应CpG-A和CpG-B。因为许多其他的病毒传感器，包括RIG-I喜欢解旋酶（RIG-1、LGP 2、MDA-5）和Dicer都属于DExD/H解旋酶家族，本研究将验证我们的中心假设：1）DExD/H-box解旋酶DHX 36和DHX 9可能代表pDC中新的TLR 9非依赖性DNA传感器; 2）DExD/H解旋酶家族（59个成员）在抗病毒天然免疫中的作用可能比以前认为的要广泛得多。)

DESCRIPTION (provided by applicant): During the past decade, major efforts using genomic and genetic approaches have identified two major classes of innate immune receptors for sensing microbial nucleic acids: Toll-like receptors (TLR: TLR3, 7, 8, 9) and retinoic acid-inducible gene I-like helicases (RLH: RIG-I, LGP2, MDA-5). However, genomic and genetic approaches have left a major gap in our understanding on how these receptors bind nucleic acids and whether additional receptors or coreceptors exist. We decided to open this "Black Box" by taking a biochemical approach to directly isolate and characterize microbial nucleic acid-binding proteins in human plasmacytoid dendritic cells (pDCs) by CpG-DNA pull down and mass spectrometric peptide sequencing experiments. We identified two novel members of the DExD/H-box helicase family DHX36 and DHX9 that specifically bind CpG- A and CpG-B, respectively, in human pDCs. Knocking down DHX36 expression by siRNA in a pDC cell line was associated with over 50% reduction in type 1 IFN responses and abolished IRF7 nuclear translocation induced by CpG-A and knocking down DHX9 expression was associated with a diminished TNF and IL-6 response and blocking of NF-kB p50 nuclear translocation induced by to CpG-B. The DExD/H helicase family has over 50 members. Based on these preliminary data and the fact that the other viral sensors, including RIG- I-like helicases (RIG-I, LGP2, MDA-5) and Dicer, all belong to the DExD/H helicase family, this proposal will test our central hypotheses: 1) the DExD/H-box helicases DHX36 and DHX9 may represent novel TLR9- independent DNA sensors in pDCs; and 2) the DExD/H helicase family (59 members) may play a much broader role in anti-viral innate immunity than previously thought. ) PUBLIC HEALTH RELEVANCE: By CpG-DNA pull down and mass spectrometric peptide sequencing, we identified two novel DExD/H- box helicase family members, DHX36 and DHX9, that specifically bind and respond to CpG-A and CpG-B, respectively, in human pDCs. Because many other viral sensors, including RIG-I like helicases (RIG-I, LGP2, MDA-5) and Dicer, all belong to the DExD/H helicase family, this proposal will test our central hypotheses: 1) the DExD/H-box helicases DHX36 and DHX9 may represent novel TLR9-independent DNA sensors in pDCs; and 2) DExD/H helicase family (59 members) may play a much broader role in anti-viral innate immunity than previously thought. )