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能够自然感染秀丽隐杆线虫（C. elegans）的病毒，秀丽隐杆线虫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，加上线虫易于处理，为表征完整动物中的病毒感染提供了极好的机会。 Orsay 的 +ssRNA 基因组约为 6.3 kb，具有三个开放阅读框 (ORF)，编码推定的病毒 RNA 聚合酶、病毒衣壳蛋白 (CP) 和非结构蛋白 δ。 δ ORF 还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其具有与病毒体相关的头部纤维，该纤维与感染性颗粒共价连接。我们的长期目标是获得对奥赛生命周期的全面、分子层面的了解。此前对奥赛感染领域的研究主要集中在宿主身上，因此我们从病毒的角度理解奥赛复制机制存在重大知识空白。该项目的总体目标是阐明由病毒体相关的 CP-δ 头部纤维介导的奥赛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）奥赛病毒颗粒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蛋白的抗血清可有效中和奥赛； (3) fshr-1突变体和RNAi敲低蠕虫中的奥赛感染显着减少，但可以通过在肠细胞中过表达fshr-1来挽救感染。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 头部纤维的天然奥赛病毒粒子的高分辨率结构。我们将使用冷冻电镜在感染性病毒颗粒内原位获得 CP-δ 头纤维的高分辨率结构，并检查空的奥赛颗粒的结构，以确定 CP-δ 头纤维在基因组释放中的作用。目标 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截短突变体阻断奥赛感染的能力来确定δ CTD在受体结合中的作用，并测试针对包括δ CTD在内的δ不同部分产生的抗血清的中和活性。目标 3 是探索 FSHR-1 在奥赛参赛中的功能作用。我们将测试 FSHR-1 和 Orsay 之间的直接相互作用，并确定 FSHR-1 表达对扩大 Orsay 宿主细胞向性的影响。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宿主进入的过程。我们的研究结果不仅将揭示独特的 CP-δ 头纤维的结构和功能，而且有助于进一步开发 Orsay-C。线虫系统作为研究模型。