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

Orsay是已知的唯一能够自然感染秀丽线虫的病毒，线虫是生物学研究中的关键模式生物。Orsay感染的慢性、非致命性与处理线虫的简便性相结合，为描述完整动物中的病毒感染提供了一个极好的机会。奥赛病毒基因组全长约6.3kb，有三个开放阅读框(ORF)，分别编码病毒核糖核酸聚合酶、病毒衣壳蛋白(CP)和非结构蛋白δ。δ开放阅读框也可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形成了一种五聚体纤维，它被结合到具有感染性的病毒粒子中。此外，我们还证实了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出口是重要的。与其他病毒相比，奥赛病毒的独特之处在于，它有一种与病毒粒子相关的头部纤维，这种纤维与感染颗粒共价连接。我们的长期目标是对Orsay的生命周期有一个全面的、分子的了解。以往对Orsay侵染的研究大多集中在寄主上，因此从病毒的角度对Orsay的复制机制的认识存在很大的差距。该项目的主要目标是阐明病毒粒子相关的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)对Orsay病毒粒子的二十面体平均冷冻电子显微镜(Cryo-EM)重建显示了5倍对称轴上的纤维样密度；(2)针对全长δ蛋白的抗血清能有效中和Orsay；(3)在FSHR-1突变体和RNA干扰击倒蠕虫中，Orsay的感染显著降低，但通过在肠道细胞中过表达FSHR-1可挽救感染。我们的研究计划由三个独立但相辅相成的目标组成。目标1是用CP-δ头纤维产生天然奥赛病毒粒子的高分辨率结构。我们将使用冷冻-EM技术在感染性病毒粒子内原位获得CP-δ头部纤维的高分辨率结构，并检查清空的Orsay颗粒的结构，以确定CP-δ头部纤维在基因组释放中的作用。目的2是定位负责宿主受体结合的CP-δ头纤维的功能区。我们将通过测试不同δ截断突变体对Orsay感染的阻断能力来确定δCTD在受体结合中的作用，并测试针对δ不同部分(包括δCTD)的抗血清的中和活性。目的3探讨FSHR-1在Orsay进入中的功能作用。我们将测试FSHR-1与Orsay之间的直接相互作用，并确定FSHR-1的表达在扩大Orsay的宿主细胞趋向性方面的影响。我们的研究结合了冷冻-EM、生物化学、蠕虫遗传学和生物学等实验技术，深入研究了Orsay寄主进入的过程。我们的研究结果不仅将有助于揭示独特的CP-δ头部纤维的结构和功能，而且有助于进一步开发Orsay-C。以雅致系统为研究模型。

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.