The infection mechanism of the nematode virus Orsay

项目摘要 四年前发现的奥赛是第一个也是唯一一个已知的自然感染模型的病毒 线虫种类秀丽隐杆线虫（Causophabditis elegans）（C. elegans）。奥赛有一个双节段正义RNA基因组 表达总共四种多肽，包括RNA依赖性RNA聚合酶（RdRP）、衣壳蛋白（RdRP）、 蛋白（CP）、非结构蛋白δ和由核糖体移码产生的CP-δ融合蛋白。最近 我们确定了一个奥赛病毒样颗粒的晶体结构，它显示了CP结构组织 它与β-诺达病毒有关，但与那些感染昆虫的β-诺达病毒不同。 考虑到奥赛的简单性和C.优雅的模特儿 有机体，Orsay-C. elegans能够详细研究病毒复制、病毒与宿主相互作用 并利用天然病原体在完整的生物体中宿主先天性抗病毒反应。发生奥赛感染 主要存在于肠细胞中，引起异常的肠形态。奥赛δ/CP-δ的初步研究 蛋白质已经产生了令人兴奋的结果，表明在细胞进入和非裂解性病毒释放中的潜在作用， 重组宿主细胞骨架网络。我们发现，在转基因蠕虫中过表达的δ主要是 局限于/靠近动物肠的顶膜。奥赛病毒感染的蠕虫表现出紊乱 肌动蛋白网络，与肠细胞中的顶端微绒毛相关。类似的无组织肌动蛋白网络 在转基因蠕虫中单独表达δ蛋白后观察到。重组δ蛋白折叠成 纤维状分子并与几种宿主细胞骨架蛋白共纯化。 为了阐明奥赛与C.为确保线虫宿主成功复制，两个实验室 陶毅智和钟伟伟，在结构病毒学和C. elegans遗传学，分别， 将合作确定奥赛δ/CP-δ蛋白的结构和生物活性， 基于一种新的抗病毒基因，使用高通量全基因组筛选发现线虫抗病毒基因网络 基于表型的测定。我们的研究结果可能会为感染机制提供新的线索 其他胃肠道病毒如轮状病毒、星状病毒、诺如病毒等。 具体目标1：表征奥赛δ和CP-δ蛋白的结构。我们计划确定 这两种蛋白质的结构使用X射线晶体学和cryo-EM的组合。 具体目标2：表征δ/CP-δ介导的Orsay-宿主相互作用。我们计划识别宿主蛋白质 使用co-IP与δ物理相互作用，并表征分子性质和生物学意义 在体外和体内的相互作用。 具体目标3：发现宿主抗病毒基因网络。我们将进行全基因组的抗病毒筛选 基因，进行抗病毒基因的表型表征，并使用两者绘制抗病毒途径。 计算和实验方法。

Project Summary Discovered four years ago, Orsay is the first and also the only virus known to naturally infect the model nematode species Caenorhabditis elegans (C. elegans). Orsay has a bi-segmented, plus-sense RNA genome that expresses a total of four polypeptides, including an RNA-dependent RNA polymerase (RdRP), a capsid protein (CP), a nonstructural protein δ, and a CP-δ fusion protein produced by ribosomal frameshift. Recently we determined the crystal structure of an Orsay virus-like particle, which shows a CP structural organization that is related to betanodaviruses but distinct from those insect-infecting alphanodaviruses. Considering the simplicity of Orsay and the ease of laboratory manipulation of C. elegans as a popular model organism, Orsay-C. elegans enables detailed mechanistic studies on virus replication, virus-host interaction and host innate antiviral responses in an intact organism using a natural pathogen. Orsay infection occurs primarily in intestine cells causing abnormal intestinal morphology. Our preliminary studies of the Orsay δ/CP-δ proteins have generated exciting results indicating potential roles in cell entry and also non-lytic viral release by restructuring the host cytoskeleton network. We showed that overexpressed δ in transgenic worms was mainly localized to/near the apical membrane of the animal intestine. Orsay-infected worms displayed disorganized actin network that is associated with the apical microvilli in intestinal cells. Similarly disorganized actin network was observed in transgenic worms following δ protein expression alone. Recombinant δ protein folds into a fibrous molecule and co-purifies with several host cytoskeleton proteins. To elucidate how Orsay interacts with the C. elegans host to ensure successful replication, the two laboratories of Yizhi Jane Tao and Weiwei Zhong, with expertise in structural virology and C. elegans genetics, respectively, will collaborate to determine the structure and the biological activities of the Orsay δ/CP-δ proteins and to discover the nematode antiviral gene network using high-throughput, genome-wide screens based on a novel phenotype-based assay. Findings from our study will likely shed new light on the infection mechanisms used by other gastrointestinal viruses such as rotavirus, astrovirus, norovirus, etc. Specific Aim 1: Characterize the structure of the Orsay δ and CP-δ proteins. We plan to determine the structure of both proteins using the combination of X-ray crystallography and cryo-EM. Specific Aim 2: Characterize Orsay-host interaction mediated by δ/CP-δ. We plan to identify host proteins that physically interact with δ using co-IP, and to characterize the molecular nature and biological implications of such interaction both in vitro and in vivo. Specific Aim 3: Discover host antiviral gene networks. We will carry out a genome-wide screen for antiviral genes, perform phenotypic characterization of antiviral genes, and map antiviral pathways using both computational and experimental approaches.