Evaluation of Novel DExD/H Helicases in Innate Immune Signaling

描述（由申请人提供）：I型干扰素（IFN-a/p，下文称为IFN）是刺激有效抗病毒宿主防御所必需的细胞因子家族。Toll样受体（TLR）依赖性和TLR非依赖性机制都存在于病毒感染后I型IFN的产生中。在对病毒的TLR非依赖性应答的新图像中，在病毒感染过程中产生的dsRNA或未加帽的ssRNA种类分别被细胞内解旋酶黑素瘤分化抗原[MDA]-5和视黄酸诱导基因（RIG）-I识别。该事件通过线粒体蛋白干扰素启动子刺激因子（IPS）-1激活IFN的产生，以激活IFN的产生。重要的是，我们的数据和其他人的数据表明，含有死亡结构域（DD）的衔接蛋白FADD（具有死亡结构域的Fas相关蛋白）和RIP 1（受体相互作用蛋白激酶1）也是MDA-5，RIG-I和IPS-1的最佳信号传导所必需的。尽管已经确定了RIG-I、MDA-5、IPS-1、FADD和RIP 1在对病毒感染的先天免疫应答中的重要作用，但是这些关键分子被病毒感染激活以刺激IFN基因转录的方式还不清楚。事实上，如我们下面的数据所示，几乎可以肯定存在将RIG-I和MDA-5信号传导与IPS-1、FADD和RIP-1连接/复合以介导抗病毒先天免疫应答的其他细胞分子/辅因子。因此，使用酵母双杂交筛选FADD相互作用蛋白，我们已经分离出两种新的DexD/H盒RNA解旋酶。第一种称为DDX-I，使用IPS-1作为诱饵通过双杂交筛选分离。通过使用FADD作为诱饵的类似筛选，也已经鉴定了第二解旋酶，称为Fah-1（Fadd相关解旋酶）。重要的是，我们的分析已经证实，这两种解旋酶都是细胞防御病毒感染所必需的。因此，我们假设这些解旋酶可以促进FADD依赖性先天免疫应答，并提出以下具体目的：1. DDX-I的表征和分子作用机制：我们的目的是表征DDX-I，包括阐明与IPS-1的相互作用，并评估前者分子在宿主防御病毒感染中的重要性。2.）FAH-1的表征和分子作用机制：我们的目的是评估FAH-1在FADD介导的先天免疫调节中的意义，并确定该分子在病毒控制的免疫机制中的重要性。

DESCRIPTION (provided by applicant): Type I interferons (IFN-a/p, hereafter referred to as IFN) are a family of cytokines necessary for the stimulation of effective anti-viral host defense. Both Toll Like Receptor (TLR) dependent and TLR- ndependent mechanisms exist for the production of type I IFNs following viral infection. In an emerging picture of TLR-independent responses to viruses, dsRNA or uncapped ssRNA species produced during the course of virus infection are recognized by the intracellular helicases melanoma differentiation antigen [MDA)-5 and retinoic acid inducible gene (RIG)-I, respectively. This event activates production of IFN via a mitochondrial protein, interferon promoter stimulator (IPS)-1, to activate IFN production. Importantly, our data and those of others show that the death-domain (DD)-containing adaptor proteins FADD (Fas-associated protein with death domain) and RIP1 (receptor interacting protein kinase 1) are also essential for optimal signaling by MDA-5, RIG-I and IPS-1. Although essential roles have been established for RIG-I, MDA-5, IPS-1, FADD and RIP1 in innate immune responses to virus infection, the manner in which these key molecules are activated by virus infection to stimulate IFN gene transcription are poorly defined. Indeed, as our data below indicate, there are almost certainly other cellular molecules/co-factors that connect/complex RIG-I and MDA-5 signaling to IPS-1, FADD and RIP-1 to mediate anti-viral innate immune responses. Accordingly, using a yeast two-hybrid screen for FADD-interacting proteins, we have isolated two novel DexD/H box RNA helicases. The first, referred to as DDX-I, was isolated through a two hybrid screen using IPS-1 as a bait. As second helicase, referred to as Fah-1 (for Fadd-associated helicase), has also been identified, through similar screens using FADD as bait. Importantly, our analysis has confirmed that both helicases are required for cellular defense against virus infection. We therefore hypothesize that these helicases may facilitate FADD-dependent innate immune responses, and propose the following Specific Aims: 1.) Characterization of DDX-I and molecular mechanisms of action: We aim to characterize DDX-I, including elucidating interactions with IPS-1, and evaluate the former molecule's importance in host defense against virus infection. 2.) Characterization of FAH-1 and molecular mechanisms of action: We aim to evaluate the significance of FAH-1 in FADD-mediated innate immune regulation and determine this molecules importance in immune mechanisms of viral control.