Host defense against respiratory virus infections

描述(申请人提供)：先天抗病毒免疫包括一种宿主防御机制，以限制病毒传播。宿主细胞通过称为模式识别受体(PRRs)的特殊受体识别入侵的病毒，这种受体可以特异性地识别病原体相关的分子模式(PAMP)。Toll样受体和RIG样解旋酶受体(如RigI和MDA5)识别病毒特异性PAMPs，通过激活转录因子干扰素调节因子3(IRF3)诱导抗病毒反应。IRF3的激活导致抗病毒细胞因子干扰素-α/β(IFN)的产生。最近我们发现了核苷酸结合寡聚化结构域2(NOD2)蛋白，属于核苷结合结构域(NBD)和亮氨酸富集区(LRR)家族的细胞质受体，是一种病毒PRR。我们的初步研究表明，NOD2可以在感染人类呼吸道合胞病毒(RSV具有单链RNA或单链RNA作为其基因组)后触发抗病毒反应(通过激活IRF3/干扰素)，人类呼吸道合胞病毒是导致全球婴儿、儿童和老年人肺部感染的主要病原体。因此，目前的建议是：a)建立NOD2作为一种新的病毒PRR，b)研究NOD2激活的机制，c)鉴定和鉴定激活的NOD2下游的分子，d)研究NOD2在RSV致病、RSV诱导的肺部炎症和肺部疾病发展中的体内作用。我们的初步研究表明，在RSV感染细胞中表达NOD2导致了IRF3/干扰素的激活。进一步的研究表明：1)NOD2能识别单链RNA激活IRF3/干扰素；2)线粒体外膜驻留蛋白IPS-1可能作用于NOD2下游，激活IRF3/干扰素。因此，我们推测NOD2是病毒PRR的一个新成员，可以识别病毒单链RNA基因组，在线粒体IPS-1蛋白激活后启动抗病毒反应。这一假说将通过关注以下特定目标来检验：目标1.研究NOD2在抗RSV病毒反应中的作用--这个目标的主要问题是-NOD2是否构成了一种病毒PRR，可以激活感染细胞中的IRF3/干扰素？我们将研究NOD2是否参与了呼吸道合胞病毒感染正常原代人支气管上皮(NHBE)细胞过程中IRF3/干扰素的激活。NOD2基因敲除(KO)小鼠在宿主防御RSV致病和RSV介导的肺部炎症/疾病过程中的生理学相关性也将被建立。目的#2.研究NOD2的激活机制--这个目的的主要问题是：a)NOD2识别单链RNA并与线粒体定位的IPS-1相互作用激活IRF3/干扰素吗？这个问题将通过-a)研究NOD2与单链RNA(RSV基因组和合成单链RNA)和IPS-1的关系，b)检测NOD2介导的IRF3/干扰素在缺乏IPS-1的细胞中的激活，以及c)确定参与单链RNA识别和IPS-1相互作用的特定NOD2结构域(S)。

DESCRIPTION (provided by applicant): Innate antiviral immunity comprises a host defense mechanism for restricting virus spread. The host cells recognizes invading viruses by specialized receptors known as pattern recognition receptors (PRRs) that can specifically identify pathogen associated molecular patterns (PAMPs). Toll-like receptors (TLRs) and RIG like helicase (RLH) receptors (e.g. RIGI and Mda5) are known to recognize virus specific PAMPs for induction of antiviral response mediated by activation of the transcription factors interferon regulatory factor-3 (IRF3). IRF3 activation results in production of antiviral cytokine interferon-a/¿ (IFN). Recently we identified nucleotide- binding oligomerization domain-2 (NOD2) protein, belonging to the nucleotide binding domain (NBD) and leucine-rich-region (LRR) containing family (known as NLRs) of cytoplasmic receptors as a viral PRR. Our preliminary studies have demonstrated that NOD2 can trigger an antiviral response (via activation of IRF3/IFN) following infection with human respiratory syncytial virus (RSV possess single stranded RNA or ssRNA as its genome), the major etiological viral agent causing worldwide pulmonary infections among infants, children and elderly. Therefore the current proposal is aimed at - a) establishing NOD2 as a new viral PRR, b) studying the mechanism of NOD2 activation, c) identifying and characterizing the molecules that act downstream of activated NOD2, and d) investigating the in vivo role of NOD2 during RSV pathogenesis, RSV induced lung inflammation and development of lung disease. Our preliminary studies demonstrated that expression of NOD2 in RSV infected cells results in activation of IRF3/IFN. Further studies revealed that - a) NOD2 can recognize ssRNA to activate IRF3/IFN, and b) mitochondrial outer membrane residing protein IPS-1 (MAVS) may act downstream of NOD2 for activation of IRF3/IFN . Thus, we hypothesize that NOD2 is a new member of viral PRR that can recognize viral ssRNA genome to launch an antiviral response following activation of mitochondrial IPS-1 protein. The hypothesis will be tested by focusing on the following specific aims: Aim # 1. Study the role of NOD2 during antiviral response against RSV - The major question of this aim is - Does NOD2 constitute a viral PRR that can activate IRF3/IFN in infected cells? We will investigate whether NOD2 is involved in IRF3/IFN activation during RSV infection of normal primary human bronchial epithelial (NHBE) cells. The physiological relevance of NOD2 during host defense against RSV pathogenesis and RSV mediated lung inflammation/disease will also be established by using NOD2 knock-out (KO) mice. Aim # 2. Study the mechanism of NOD2 activation - The major question of this aim is - a) Does NOD2 recognizes ssRNA and interacts with mitochondrial localized IPS-1 to activate IRF3/IFN? This question will be addressed by - a) studying association of NOD2 with ssRNA (RSV genome and synthetic ssRNA) and IPS-1, b) examining NOD2 mediated IRF3/IFN activation in cells lacking IPS-1, and c) identifying the specific NOD2 domain(s) involved in ssRNA recognition and IPS-1 interaction.