The infection mechanism of the nematode virus Orsay

线虫病毒奥赛的感染机制

• 批准号: 9196930
• 负责人: Yizhi Jane Tao
• 金额: $ 37.84万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-15 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9196930
• 关键词: Actins; Animal Model; Animals; Antiviral Agents; Antiviral Response; Apical; Astrovirus; Biological; Biological Assay; Biological Process; Caenorhabditis elegans; Capsid; Capsid Proteins; Cells; Chimeric Proteins; Chromosome Mapping; Cytoskeleton; Defense Mechanisms; Ensure; Fishes; Flavivirus; Genes; Genetic; Genome; Human; In Vitro; Infection; Insecta; Internet; Intestines; Laboratories; Lead; Life; Light; Longevity; Mammals; Marines; Mediating; Modeling; Molecular; Morphology; Natural Immunity; Nature; Necrosis; Nematoda; Nonstructural Protein; Norovirus; Organism; Pathway interactions; Phenotype; Phylogenetic Analysis; Process; Proteins; RNA; RNA-Directed RNA Polymerase; Recombinants; Replication-Associated Process; Research; Ribosomal Frameshifting; Role; Rotavirus; Structure; System; Transgenic Organisms; Translations; Vaccinia virus; Viral; Virus; Virus Diseases; Virus Replication; Virus-like particle; X-Ray Crystallography; apical membrane; base; cellular microvillus; delta protein; gastrointestinal; genome-wide analysis; in vivo; in vivo Model; novel; overexpression; particle; pathogen; polypeptide; protein expression; protein folding; virology; virus host interaction

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.

项目摘要 四年前发现的奥赛病毒是第一个也是唯一已知的自然感染该模型的病毒 线虫物种秀丽线虫(C.elegans)奥赛有一个双片段正义rna基因组。 它总共表达四种多肽，包括依赖于RNA的RNA聚合酶(Rdrp)、衣壳 蛋白质(CP)是一种非结构蛋白δ，是核糖体移码产生的CP-δ融合蛋白。最近 我们测定了一种类似奥赛病毒的颗粒的晶体结构，它显示出CP结构 这与Betanoda病毒有关，但与那些感染昆虫的Alphanoda病毒不同。 考虑到Orsay的简单性和实验室操作线虫的简便性，这是一个流行的模型 Orsay-C.Elgans能够对病毒复制、病毒与宿主的相互作用进行详细的机制研究 并使用自然病原体在完整的有机体中进行先天抗病毒反应。奥赛感染发生 主要发生在肠道细胞内，导致肠道形态异常。Orsayδ/CP-δ的初步研究 蛋白质产生了令人兴奋的结果，表明了在细胞进入和非裂解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 位于/靠近动物肠道的顶膜。感染Orsay的蠕虫表现出杂乱无章 与肠道细胞顶端微绒毛相关的肌动蛋白网络。肌动蛋白网络同样杂乱无章 在仅表达δ蛋白的转基因蠕虫中观察到。重组δ蛋白折叠成一个 纤维状分子，与几种宿主细胞骨架蛋白共同纯化。 为了阐明奥赛如何与线虫宿主相互作用以确保成功复制，这两个实验室 分别在结构病毒学和线虫遗传学方面有专长的陶以智和钟伟伟， 将合作确定Orsayδ/CP-δ蛋白的结构和生物活性，并 利用高通量全基因组筛选发现线虫抗病毒基因网络基于一种新的 基于表型的分析。我们的研究结果可能会为我们所使用的感染机制提供新的线索 由其他胃肠道病毒感染，如轮状病毒、星状病毒、诺如病毒等。 具体目标1：鉴定Orsayδ和CP-δ蛋白的结构。我们计划确定 用X射线结晶学和低温电子显微镜相结合的方法研究这两种蛋白质的结构。 具体目标2：鉴定δ/CP-δ介导的Orsay-宿主相互作用。我们计划鉴定宿主蛋白 利用共IP与δ进行物理相互作用，并表征其分子性质和生物学意义 在体外和体内的这种相互作用。 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查 基因，执行抗病毒基因的表型特征，并使用两者绘制抗病毒途径 计算方法和实验方法。