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: 10511348
• 负责人: Yizhi Jane Tao
• 金额: $ 22.77万
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10511348
• 关键词: Animal Model; Animals; Apical; Binding; Biochemistry; Biological Assay; Biology; C-terminal; Caenorhabditis elegans; Capsid; Capsid Proteins; Cells; Chimeric Proteins; Chronic; Cryoelectron Microscopy; Crystallization; 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; Light; 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; Structure; Surface; System; Techniques; Testing; Tropism; Viral; Viral Genome; Virion; Virus; Virus Diseases; Work; biological research; delta protein; density; experimental study; gene product; knock-down; 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.

奥赛病毒是唯一已知的能够自然感染秀丽隐杆线虫（C. elegans）的病毒，而奥赛感染是生物学研究中的关键模型生物，奥赛感染的慢性、非致命性加上易于处理的秀丽隐杆线虫提供了绝佳的机会。 Orsay 具有约 6.3 kb 的 +ssRNA 基因组，具有三个编码假定的病毒 RNA 聚合酶（病毒衣壳）的开放阅读框 (ORF)。 δ ORF 也可以通过核糖体移码表达为 CP-δ 融合蛋白。我们实验室的工作表明，CP-δ 形成五聚体纤维，并融入感染性病毒颗粒中。此外，我们已经确定 CP-δ 头部纤维介导受体结合和宿主进入，而游离 δ 蛋白对于非裂解性病毒流出很重要，与其他病毒相比，奥赛病毒的独特之处在于具有我们的长期目标是对奥赛生命周期进行全面的分子了解，该领域的奥赛感染研究主要集中在宿主身上。从病毒的角度来看，我们对 Orsay 复制机制的理解存在重大知识差距。该项目的总体目标是阐明由病毒颗粒相关的 CP-δ 头部纤维介导的 Orsay 宿主进入的分子基础。支持通过强有力的初步数据：（1）奥赛病毒粒子的二十面体平均冷冻电子显微镜（cryo-EM）重建显示在5倍对称轴处有纤维状密度；（2）针对全长δ蛋白的抗血清可以(3) fshr-1突变体和RNAi敲除蠕虫中的Orsay感染显着减少，但感染可以通过以下方法挽救：我们的研究计划由三个独立但互补的目标组成，目标 1 是生成具有 CP-δ 头部纤维的高分辨率结构。目标 2 是绘制感染性病毒粒子内 CP-δ 头纤维原位的高分辨率结构，并检查空奥赛颗粒的结构以确定 CP-δ 头纤维在基因组释放中的作用。 CP-δ 头纤维负责宿主受体结合的功能区域 我们将通过测试不同 δ 截短突变体阻断 Orsay 感染的能力来确定 δ CTD 在受体结合中的作用，并测试针对其产生的抗血清的中和活性。 δ 的不同部分，包括 δ CTD 目标 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 的影响。 FSHR-1在扩大奥赛宿主细胞向性中的表达我们的研究结合了冷冻电镜、生物化学、蠕虫遗传学和生物学等实验技术来彻底研究奥赛进入宿主的过程。阐明了独特的 CP-δ 头部纤维的结构和功能，同时也有助于进一步开发 Orsay-C 线虫系统作为研究模型。