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

奥赛病毒是目前已知的唯一能够自然感染秀丽隐杆线虫（C. habditis elegans）的病毒。elegans），生物学研究中的关键模式生物。奥赛感染的慢性、非致命性以及C.秀丽隐杆线虫提供了一个极好的机会来表征完整动物中的病毒感染。奥赛具有约6.3 kb的+ssRNA基因组，具有编码推定的病毒RNA聚合酶、病毒衣壳蛋白（CP）和非结构蛋白δ的三个开放阅读框（ORF）。δ ORF也可以通过核糖体移码表达为CP-δ融合蛋白。我们实验室的工作显示，CP-δ形成了一种五聚体纤维，它被整合到感染性病毒体中。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外出是重要的。与其他病毒相比，奥赛病毒的独特之处在于它有一个与病毒体相关的头部纤维，该纤维与感染性颗粒共价连接。我们的长期目标是对奥赛的生命周期有一个全面的、分子的了解。以前关于奥赛感染的研究主要集中在宿主上，因此我们从病毒的角度对奥赛复制机制的理解存在很大的知识差距。该项目的首要目标是阐明由病毒体相关CP-δ头纤维介导的奥赛宿主进入的分子基础。初步研究结果表明：（1）奥赛病毒粒子的二十面体平均冷冻电镜（cryo-EM）重建显示在5重对称轴上呈纤维状密度;（2）针对全长δ蛋白的抗血清可以有效地中和奥赛病毒粒子;（3）在fshr-1突变体和RNAi敲低蠕虫中，奥赛感染显著降低，但在肠细胞中过表达fshr-1可以挽救感染。我们的研究计划包括三个独立但互补的目标。目的1是用CP-δ头纤维生成天然奥赛病毒粒子的高分辨率结构。我们将使用cryo-EM在感染性病毒体中原位获得CP-δ头纤维的高分辨率结构，并检查清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。目的二是定位CP-δ头纤维与宿主受体结合的功能区。我们将通过测试不同δ截短突变体阻断奥赛感染的能力，并测试针对δ的不同部分（包括δ CTD）产生的抗血清的中和活性，来确定δ CTD在受体结合中的作用。目的3探讨FSHR-1在奥赛进入中的功能作用。我们将测试FSHR-1和奥赛之间的直接相互作用，并确定FSHR-1表达在扩展奥赛的宿主细胞嗜性中的影响。我们的研究使用了实验技术，包括冷冻EM，生物化学，蠕虫遗传学和生物学的组合，彻底调查的过程中，奥赛主机进入。本研究结果不仅有助于进一步了解CP-δ头纤维的结构和功能，而且有助于进一步开发Orsay-C。elegans系统作为研究模型。

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.