Evaluation of Novel DExD/H Helicases in Innate Immune Signaling

说明(申请人提供)：I型干扰素(干扰素-a/p，以下简称干扰素)是刺激有效的抗病毒宿主防御所必需的细胞因子家族。Toll样受体(TLR)依赖和非TLR依赖的机制都存在于病毒感染后产生I型IFN。在TLR非依赖病毒反应的新图景中，病毒感染过程中产生的dsRNA或未封顶的单链RNA物种分别被细胞内螺旋酶黑色素瘤分化抗原[MDA]-5和维甲酸诱导基因(RIG)-I识别。该事件通过线粒体蛋白-干扰素启动子刺激物(IPS)-1激活干扰素的产生，从而激活干扰素的产生。重要的是，我们和其他人的数据表明，含有死亡结构域(DD)的适配蛋白FADD(具有死亡结构域的Fas相关蛋白)和RIP1(受体相互作用蛋白激酶1)对于MDA-5、RIG-I和IPS-1的最佳信号传递也是必不可少的。虽然RIG-I、MDA-5、IPS-1、FADD和RIP1在病毒感染的先天免疫应答中发挥了重要作用，但病毒感染激活这些关键分子以刺激干扰素基因转录的方式尚不清楚。事实上，正如我们下面的数据显示的那样，几乎可以肯定还有其他细胞分子/辅助因子连接/复杂的RIG-I和MDA-5信号转导IPS-1、FADD和RIP-1，以介导抗病毒的先天免疫反应。因此，利用酵母双杂交筛选FADD相互作用蛋白，我们分离出两个新的DexD/H盒RNA解旋酶。第一种被称为DDX-I，是通过使用IPS-1作为诱饵的双混合屏幕分离出来的。作为第二解旋酶，称为Fah-1(FADD相关解旋酶)，也已通过以FADD为诱饵的类似筛选被鉴定。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能促进FADD依赖的先天免疫反应，并提出以下具体目标：1)DDX-I的特性和分子作用机制：我们的目标是描述DDX-I的特性，包括阐明与IPS-1的相互作用，并评估前者在宿主防御病毒感染中的重要性。2.)FAH-1的特性和分子作用机制：我们的目的是评估FAH-1在FADD介导的天然免疫调节中的意义，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.