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

Orsay是唯一已知的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式生物。奥赛感染的慢性、非致死性与秀丽隐杆线虫易于处理相结合，为在完整动物中表征病毒感染提供了极好的机会。Orsay的+ssRNA基因组约6.3 kb，有三个开放阅读框（orf）编码假定的病毒RNA聚合酶、病毒衣壳蛋白（CP）和非结构蛋白δ。δ ORF也可以通过核糖体移码表达为CP-δ融合蛋白。我们实验室的研究表明，CP-δ形成了一种五聚体纤维，可被纳入感染性病毒粒子。此外，我们已经确定CP-δ头纤维介导受体结合和宿主进入，而自由δ蛋白对于非裂解性病毒的输出很重要。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粒子相关的头部纤维，它以共价的方式附着在感染颗粒上。我们的长期目标是获得对奥赛生命周期的全面的分子理解。以往在该领域对Orsay感染的研究主要集中在宿主上，因此从病毒的角度对Orsay复制机制的理解存在很大的知识缺口。本项目的总体目标是阐明由病毒粒子相关的CP-δ头部纤维介导的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)奥赛病毒粒子的二十面体平均冷冻电镜（cro - em）重建显示在5倍对称轴上有纤维状密度；(2)针对全长δ蛋白的抗血清能有效中和Orsay；(3) fshr1突变体和RNAi敲除虫的Orsay感染明显减少，但在肠细胞中过表达fshr1可挽救感染。我们的研究计划由三个独立但又相辅相成的目标组成。目的1是利用CP-δ头纤维生成天然奥赛病毒粒子的高分辨率结构。我们将使用冷冻电镜（cryo-EM）在感染性病毒粒子内原位获得CP-δ头纤维的高分辨率结构，并检测空化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CP-δ头纤维负责宿主受体结合的功能区。我们将通过测试不同δ截断突变体阻断Orsay感染的能力来确定δ CTD在受体结合中的作用，并测试抗血清对δ的不同部分（包括δ CTD）的中和活性。目的3是探讨fshr1在Orsay入口中的功能作用。我们将测试fshr1和Orsay之间的直接相互作用，并确定fshr1表达对扩大Orsay宿主细胞向性的影响。我们的研究采用低温电镜、生物化学、蠕虫遗传学和生物学等实验技术相结合，深入研究了奥赛寄主的进入过程。我们的研究结果不仅有助于揭示独特的CP-δ头纤维的结构和功能，而且有助于进一步开发Orsay-C。秀丽隐杆线虫系统作为研究模型。

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.