Host defense against respiratory virus infections

描述（由申请方提供）：天然抗病毒免疫包括限制病毒传播的宿主防御机制。宿主细胞通过称为模式识别受体（PRR）的专门受体识别入侵病毒，所述PRR可以特异性地识别病原体相关分子模式（PAMP）。已知Toll样受体（TLR）和RIG样解旋酶（RLH）受体（例如RIGI和Mda 5）识别病毒特异性PAMP，用于诱导由转录因子干扰素调节因子-3（IRF 3）的活化介导的抗病毒应答。IRF 3激活导致抗病毒细胞因子干扰素-α（IFN）的产生。最近，我们鉴定了核苷酸结合寡聚化结构域-2（NOD 2）蛋白，其属于含有核苷酸结合结构域（NBD）和亮氨酸富集区（LRR）的胞质受体家族（称为NLR），作为病毒PRR。我们的初步研究已经证明，NOD 2可以在感染人呼吸道合胞病毒（RSV具有单链RNA或ssRNA作为其基因组）后触发抗病毒应答（通过激活IRF 3/IFN），所述人呼吸道合胞病毒是引起婴儿、儿童和老年人的全球肺部感染的主要病原病毒剂。因此，本发明的目的在于- a）建立NOD 2作为新的病毒PRR，B）研究NOD 2活化的机制，c）鉴定和表征在活化的NOD 2下游起作用的分子，和d）研究NOD 2在RSV发病机制、RSV诱导的肺部炎症和肺部疾病发展过程中的体内作用。我们的初步研究表明，在RSV感染的细胞中NOD 2的表达导致IRF 3/IFN的激活。进一步的研究表明：a）NOD 2可以识别ssRNA激活IRF 3/IFN，B）线粒体外膜驻留蛋白IPS-1（MAVS）可能在NOD 2下游起作用，激活IRF 3/IFN。因此，我们假设NOD 2是病毒PRR的新成员，可以识别病毒ssRNA基因组，在线粒体IPS-1蛋白激活后启动抗病毒反应。将通过关注以下具体目标来检验假设：目标1。研究NOD 2在抗RSV抗病毒反应中的作用-该目标的主要问题是-NOD 2是否构成病毒PRR，可以激活感染细胞中的IRF 3/IFN？我们将调查是否NOD 2参与IRF 3/IFN激活在RSV感染的正常原代人支气管上皮细胞（NHBE）。还将通过使用NOD 2敲除（KO）小鼠来建立在宿主防御RSV发病机制和RSV介导的肺部炎症/疾病期间NOD 2的生理相关性。目标二。研究NOD 2激活的机制-该目的的主要问题是- a）NOD 2是否识别ssRNA并与线粒体定位的IPS-1相互作用以激活IRF 3/IFN？该问题将通过以下方式解决：a）研究NOD 2与ssRNA（RSV基因组和合成ssRNA）和IPS-1的关联，B）在缺乏IPS-1的细胞中检查NOD 2介导的IRF 3/IFN活化，以及c）鉴定参与ssRNA识别和IPS-1相互作用的特异性NOD 2结构域。

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.