Elucidation of novel pathways components of C. elegans viral immunity

这个研究项目代表了我实验室的一个新方向，即利用秀丽隐杆线虫作为模型生物，从我实验室本科生获得的初步数据中发现涉及病毒先天免疫的新途径。感兴趣的途径目前在蠕虫中尚不清楚，但在脊椎动物中存在。他们在秀丽隐杆线虫中的发现将为我们对这种模式生物的认识提供一个重要的进展，使我们对进化过程中这些过程的保护有了新的认识，因此，它们在脊椎动物中的重要性。秀丽隐杆线虫的先天免疫系统在区分非致病性和致病性细菌方面非常有效。但它的基因组不包含与哺乳动物感染病毒免疫反应有关的重要途径成分。在脊椎动物中，先天免疫反应是由识别病毒核酸的特异性受体（称为病原体识别受体（Pathogens Recognition receptors， PRR））介导的。这些受体通过称为寡核苷酸结合域（OB）的结合域识别病毒核酸。在感染过程中，这些PRR识别病毒的核酸，并因此发生一系列信号事件来上调抗病毒机制。在秀丽隐杆线虫中，这些PRR以及激活病毒免疫反应的分子成分是未知的，并且秀丽隐杆线虫中OB折叠家族的一些成员是否为PRR仍有待发现。我们已经在我的实验室建立了秀丽隐杆线虫感染系统的dsRNA奥赛病毒。我们现在可以使用为这种模式生物广泛开发的反向遗传学工具，从中我们可以获得许多试剂，以确定被这种病毒激活的途径成分。长期：我们的长期目标是确定模式生物秀丽隐杆线虫中奥赛病毒感染激活的未知病毒免疫途径的分子成分。我们可能会揭示一种古老的dsRNA抗病毒免疫途径，目前尚不清楚，可能在动物中保守。短期：当前的项目可以用两个较短的目标来描述。(i)确定OB折叠在感染诺达病毒的秀丽隐杆线虫中的作用。哪些OB折叠是触发秀丽隐杆线虫免疫反应的奥赛病毒核酸的模式识别受体。它们存在吗？我们将使用OB折叠敲低株和/或过表达OB基因启动子或编码区的转基因系来确定病毒是否干扰其中一些基因的基因表达。（ii）确定奥赛病毒感染后哪些抗病毒宿主效应物因途径激活而受到调节。我们将使用RNA测序技术来分析感染和非感染菌株之间差异表达的基因。影响：目标1中提出的研究可能会确定在秀丽隐杆线虫中增加病毒免疫反应的未知模式识别受体。识别它们是对这种生物的基础知识的一个重要进步。目标2将深入了解秀丽隐杆线虫如何对病毒感染产生先天免疫反应，尽管已知的脊椎动物途径成分很少。这种新型信号级联的组成部分的鉴定具有在脊椎动物中发现新型抗病毒效应器和调节剂的真正潜力。病毒先天免疫反应的这些新特征可能是一种在脊椎动物中进化保守的古老机制。对这些效应物和调节物的进一步研究可能会导致新的抗病毒和抗炎疗法的候选靶点，以及可能在脊椎动物中缺失的新的感染生物标志物。

This research program represents a new direction in my laboratory towards the discovery of novel pathways involved in viral innate-immunity using C. elegans as a model organism that build from preliminary data obtained by undergraduate students in my laboratory. The pathways of interest are currently unknown in the worm, but exist in vertebrates. Their discovery in C. elegans would provide an important advance in knowledge in this model organism that would give us novel insights into the conservation of these processes during evolution and therefore, their importance in vertebrates. The C. elegans innate immune system is remarkably efficient at distinguishing non-pathogenic from pathogenic bacteria. But its genome does not contain the important pathway components that are involved in viral immune response for infection in mammalian. In vertebrates the innate immune response is mounted by specific receptors that recognize viral nucleic-acids called Pathogens Recognition Receptors (PRR). These receptors recognize viral nucleic acids by a binding domain called Oligonucleotide Binding domain (OB). During infection these PRR recognize viral nucleic-acids and as a result a series of signaling events take place to up regulate anti-viral mechanisms. In C. elegans these PRR’s as well as the molecular components that activate the viral immune response are unknown and it remains to be discovered if some members of the OB fold family in C. elegans are PRR. We have established in my laboratory the C. elegans infection system by the dsRNA Orsay virus. We can now use reverse genetics tools that have been extensively developed for this model organism and from which many reagents can be made available to us to identify the pathways components activated by this virus. Long term: Our long term goal is to identify the molecular components of the unknown viral immunity pathways activated by orsay virus infection in the model organism C. elegans. We may reveal an ancient dsRNA antiviral immune pathway that is currently unknown and possibly conserved in animals. Short term: The current program can be described by two shorter objectives. (i) Determine the role of the OB fold in C. elegans infected by nodavirus orsay. Which OB fold are the pattern recognition receptors of the orsay-virus nucleic-acids that trigger the immune response in C. elegans . Do they exist? We will use OB fold knockdown strains and/or transgenic line over-expressing OB gene promotor or coding region to determine if the virus interfere with gene expression for some of these genes. (ii) Determine which antiviral host effectors are regulated as a result of pathway activation after orsay virus infection. We will use RNA sequencing techniques to profile differentially expressed genes between infected and non infected strains. Impact : The proposed research in objective 1 may identify the unknown Pattern Recognition Receptor(s) that are mounting the viral immune response in C. elegans. Identifying them is an important advance in basic knowledge of this organism. Objective 2 will give insight into how C. elegans mounts the innate immune response against viral infection, despite having few of the known vertebrate pathway components. The identification of components of such a novel signaling cascade has real potential for the discovery of novel antiviral effectors and regulators in vertebrates. These novel features of viral innate immune response may be an ancient mechanism evolutionarily conserved within vertebrates. Further studies on these effectors and regulators could lead to candidate targets for novel antiviral and anti-inflammatory therapies as well as new biomarkers for infection that may have been missed in vertebrates.