The molecular basis of Orsay virus entry mediated by the CP-delta head fiber

CP-delta头纤维介导奥赛病毒进入的分子基础

• 批准号: 10622529
• 负责人: Yizhi Jane Tao
• 金额: $ 19.21万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622529
• 关键词: Animals; Apical; Binding; Biochemistry; Biological Assay; Biology; C-terminal; Caenorhabditis elegans; Capsid; Capsid Proteins; Cells; Chimeric Proteins; Chronic; Cryoelectron Microscopy; DNA-Directed RNA Polymerase; Data; Distal; Engineering; Fiber; Filament; Food; Genetic; Genome; Goals; Head; Image; Immune Sera; In Situ; Infection; Internet; Intestines; Knowledge; Length; Life Cycle Stages; Longevity; Maps; Mediating; Modeling; Molecular; Molecular Conformation; N-terminal; Nature; Nematoda; Nematode infections; Neutralization Tests; Nonlytic; Nonstructural Protein; Open Reading Frames; Pathway interactions; Play; Process; RNA; RNA Interference; RNA-Directed RNA Polymerase; Research; Resolution; Ribosomal Frameshifting; Role; Shapes; Structure; Surface; System; Techniques; Testing; Tropism; Viral; Viral Genome; Virion; Virus; Virus Diseases; Work; biological research; delta protein; experimental study; gene product; knock-down; model organism; mutant; overexpression; particle; pathogen; practical application; receptor; receptor binding; reconstruction; uptake; viral RNA; virus host interaction

Orsay is the only known virus capable of naturally infecting Caenorhabditis elegans (C. elegans), a key model organism in biological research. The chronic, non-lethal nature of Orsay infection combined with the ease of handling C. elegans provides an excellent opportunity to characterize virus infection in an intact animal. Orsay has a +ssRNA genome of ~6.3 kb with three open reading frames (ORFs) encoding the putative viral RNA polymerase, the viral capsid protein (CP), and a nonstructural protein δ. The δ ORF can also be expressed as a CP-δ fusion protein through ribosomal frameshift. Work from our lab revealed that CP-δ forms a pentameric fiber that is incorporated into the infectious virion. In addition, we have established that the CP-δ head fiber mediates receptor binding and host entry, while the free δ protein is important for nonlytic viral egress. Compared to other viruses, Orsay is unique in having a virion-associated head fiber that is covalently attached to the infectious particle. Our long-term goal is to obtain a comprehensive, molecular understanding of the Orsay life cycle. Previous research on Orsay infection in the field has mostly focused on the host, and therefore there is a major knowledge gap in our understanding of Orsay replication mechanisms from the viral standpoint. The overarching goals of this project are to elucidate the molecular basis of Orsay host entry mediated by the virion- associated CP-δ head fiber. Our proposed research is supported by strong preliminary data: (1) an icosahedrally averaged cryo-electron microscopy (cryo-EM) reconstruction of the Orsay virion shows fiber-like densities at 5- fold symmetry axes; (2) antisera raised against the full-length δ protein can effectively neutralize Orsay; (3) Orsay infection was significantly reduced in fshr-1 mutants and RNAi knock-down worms, but infection could be rescued by overexpressing fshr-1 in intestinal cells. Our research plan consists of three independent but complementary aims. Aim 1 is to generate a high-resolution structure of native Orsay virions with the CP-δ head fiber. We will use cryo-EM to obtain a high-resolution structure of the CP-δ head fiber in situ within the infectious virion, and examine the structure of emptied Orsay particles to determine the role of the CP-δ head fiber in genome release. Aim 2 is to map functional regions of the CP-δ head fiber responsible for host receptor binding. We will determine the role of the δ CTD in receptor binding by testing the ability of different δ truncation mutants to block Orsay infection, and test the neutralization activity of antisera raised against different parts of δ including the δ CTD. Aim 3 is to explore the functional role of FSHR-1 in Orsay entry. We will test for direct interactions between FSHR-1 and Orsay, and determine the impact of FSHR-1 expression in expanding the host cell tropism of Orsay. Our research uses a combination of experimental techniques including cryo-EM, biochemistry, worm genetics, and biology to thoroughly investigate the process of Orsay host entry. Results from our research will not only shed light on the structure and function of the unique CP-δ head fiber but also contribute to further developing the Orsay-C. elegans system as a research model.

奥赛（Orsay）是唯一能够自然感染的秀丽隐杆线虫（秀丽隐杆线虫）的已知病毒，这是生物学研究中的关键模型生物。奥赛感染的慢性，非致命的性质结合了秀丽隐杆线虫的易于处理，为表征完整动物的病毒感染提供了绝佳的机会。 Orsay的A +SSRNA基因组为约6.3 kb，带有三个编码假定病毒RNA聚合酶，病毒capsid蛋白（CP）和非结构蛋白δ的开放式阅读框（ORF）。 δORF也可以通过核糖体框架来表示为CP-δ融合蛋白。我们实验室的工作表明，CP-δ形成了五个五纤维，并掺入传染性病毒中。此外，我们已经确定CP-δ头纤维介导受体结合和宿主进入，而游离δ蛋白对于非散热病毒出口很重要。与其他病毒相比，Orsay在具有共价附着在传染性颗粒上的病毒体相关的头纤维方面是独一无二的。我们的长期目标是获得对奥赛生命周期的全面，分子的理解。以前关于该领域奥赛感染的研究主要集中在宿主上，因此，从病毒的角度来看，我们对奥赛复制机制的理解存在很大的知识差距。该项目的总体目标是阐明由病毒体相关的CP-δ头纤维介导的Orsay宿主进入的分子基础。我们提出的研究得到了强有力的初步数据的支持：（1）Orsay Virion的二十二个平均冷冻电子显微镜（Cryo-EM）重建显示，在5倍对称轴上显示了纤维样密度； （2）针对全长δ蛋白饲养的抗血清可以有效地中和Orsay； （3）在FSHR-1突变体和RNAi敲低蠕虫中，Orsay感染显着降低，但可以通过过表达肠细胞中的FSHR-1来挽救感染。我们的研究计划包括三个独立但完全的目标。目的1是用CP-δ头纤维生成天然Orsay病毒的高分辨率结构。我们将使用冷冻EM在感染性病毒体内的原位获得CP-δ头纤维的高分辨率结构，并检查空的Orsay颗粒的结构，以确定CP-δ头纤维在基因组释放中的作用。目的2是绘制负责宿主受体结合的CP-δ头纤维的功能区域。我们将通过测试不同δ截断突变体阻止Orsay感染的能力，并测试针对包括δCTD（ΔCTD）不同部分的抗血清的中和活性来确定δCTD在受体结合中的作用。 AIM 3是探索FSHR-1在Orsay条目中的功能作用。我们将测试FSHR-1和Orsay之间的直接相互作用，并确定FSHR-1表达在扩展Orsay的宿主细胞对流中的影响。我们的研究结合了包括冷冻EM，生物化学，蠕虫遗传学和生物学在内的实验技术的组合来彻底研究Orsay宿主进入的过程。我们研究的结果不仅会阐明独特的CP-δ头纤维的结构和功能，而且还有助于进一步开发Orsay-C。秀丽隐杆线系统作为研究模型。