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

描述(申请人提供)：在过去的十年里，使用基因组和遗传学方法的主要工作已经确定了两类主要的天然免疫受体来感应微生物核酸：Toll样受体(TLR：TLR3，7，8，9)和维甲酸诱导的基因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与核酸结合以及是否存在额外的受体或辅助受体的理解上留下了很大的空白。我们决定打开这个“黑匣子”，采用生化方法，通过CpG-DNA下拉和质谱肽测序实验，直接分离和鉴定人浆细胞样树突状细胞(PDCs)中的微生物核酸结合蛋白。我们鉴定了DExD/H-box解旋酶家族DHX36和DHX9的两个新成员，它们分别与人pDC中的CpG-A和CpG-B特异结合。在PDC细胞中，siRNA下调DHX36的表达可使I型干扰素应答减少50%以上，并可消除CpG-A诱导的IRF7核转位；下调DHX9的表达与降低肿瘤坏死因子和IL-6的应答以及阻断CpG-B诱导的核转位有关。DExD/H解旋酶家族有50多个成员。基于这些初步数据以及其他病毒传感器，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，这一提议将检验我们的中心假设：1)DExD/H-box解旋酶DHX36和DHX9可能代表pDC中不依赖TLR9的新的DNA传感器；以及2)DExD/H解旋酶家族(59个成员)在抗病毒天然免疫中可能发挥比先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，我们鉴定了两个新的DExD/H-box解旋酶家族成员DHX36和DHX9，它们分别与人pDC中的CpG-A和CpG-B特异结合和反应。由于许多其他病毒传感器，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，因此本提案将检验我们的核心假设：1)DExD/H-box解旋酶DHX36和DHX9可能代表pDC中不依赖TLR9的新的DNA传感器；以及2)DExD/H解旋酶家族(59个成员)可能在抗病毒天然免疫中发挥比先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. )