Elucidation of novel pathways components of C. elegans viral immunity

这项研究计划代表了我的实验室的一个新方向，即利用线虫作为模式生物，根据我实验室本科生获得的初步数据，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是未知的，但在脊椎动物中存在。它们在线虫中的发现将为这一模式生物的知识提供重要的进步，这将使我们对这些过程在进化过程中的保守以及它们在脊椎动物中的重要性有新的见解。秀丽线虫的天然免疫系统在区分非致病细菌和致病细菌方面非常有效。但它的基因组不包含哺乳动物感染病毒免疫反应所涉及的重要途径成分。在脊椎动物中，先天免疫反应是由识别病毒核酸的特定受体启动的，称为病原体识别受体(PRR)。这些受体通过一个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识别病毒核酸，结果发生了一系列信号事件，上调了抗病毒机制。在线虫中，这些PRR以及激活病毒免疫反应的分子成分尚不清楚，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由dsRNA Orsay病毒感染线虫的系统。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，从中可以获得许多试剂来鉴定这种病毒激活的途径成分。长期目标：我们的长期目标是在模式生物线虫中鉴定由奥赛病毒感染激活的未知病毒免疫途径的分子成分。我们可能会揭示一条古老的dsRNA抗病毒免疫途径，目前尚不清楚，可能在动物身上保守。短期：当前的计划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Orsay的线虫中的作用。在线虫中，哪些OB折叠是触发线虫免疫反应的奥赛病毒核酸的模式识别受体。他们真的存在吗？我们将使用OB折叠敲除菌株和/或过度表达OB基因启动子或编码区的转基因株来确定病毒是否干扰其中一些基因的基因表达。(Ii)确定哪些抗病毒宿主效应物在奥赛病毒感染后由于途径激活而受到调控。我们将使用核糖核酸测序技术来分析感染和未感染菌株之间的差异表达基因。影响：目标1中提出的研究可能会识别在线虫中装载病毒免疫反应的未知模式识别受体(S)。识别它们是对这种有机体基础知识的重要进步。目标2将深入了解线虫如何建立针对病毒感染的先天免疫反应，尽管它几乎没有已知的脊椎动物途径组件。这种新的信号级联的成分的鉴定对于在脊椎动物中发现新的抗病毒效应和调节因子具有真正的潜力。病毒先天免疫反应的这些新特征可能是脊椎动物进化中保守的一种古老机制。对这些效应器和调节器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遗漏的感染的新生物标记物。

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.