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

描述（由申请人提供）：在过去的十年中，使用基因组学和遗传学方法的主要工作已经确定了两大类用于检测微生物核酸的天然免疫受体：toll样受体（TLR: TLR3, 7,8,9）和视黄酸诱导的基因i样解旋酶（RLH: RIG-I, LGP2, MDA-5）。然而，基因组和遗传方法在我们对这些受体如何结合核酸以及是否存在其他受体或辅助受体的理解方面留下了重大空白。我们决定打开这个“黑匣子”，采用生化方法，通过CpG-DNA拉下和质谱肽测序实验，直接分离和表征人浆细胞样树突状细胞（pDCs）中的微生物核酸结合蛋白。我们发现了DExD/H-box解旋酶家族的两个新成员DHX36和DHX9，它们分别在人类pDCs中特异性结合CpG- A和CpG- b。在pDC细胞系中，通过siRNA敲低DHX36表达与CpG-A诱导的1型IFN反应降低50%以上相关，并消除了CpG-A诱导的IRF7核易位，而敲低DHX9表达与CpG-B诱导的TNF和IL-6反应降低以及NF-kB p50核易位阻断相关。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- i, LGP2, 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DHX36和DHX9可能代表pDCs中新的不依赖TLR9的DNA传感器；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。

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. )