Human 3D neuro-muscular assembloids to study cell tropism and host factor utilization of divergent neuropathogenic enteroviruses

项目摘要 肠道病毒是儿童病毒性脑膜炎和最近暴发的新兴非脊髓灰质炎的主要原因 肠道病毒（NPEV）与一种名为急性弛缓性脊髓炎（AFM）的脊髓灰质炎样瘫痪有关。 对神经发病机制至关重要的细胞成分的发现和表征有望为 揭示了治疗肠道病毒病的新方法。近年来，已经鉴定了多种受体， 对于EV-A71和EV-D 68，NPEV，其最常与AFM相关。使用无偏基因组- 在大规模筛选中，我们已将磷脂酶PLA 2G 16确定为立即起作用的进入因子 NPEV感染后受体结合的下游。多种受体和PLA 2G 16如何工作 然而，在很大程度上是未知的。感染 存在于中枢神经系统中的细胞类型对于发展严重的神经系统形式至关重要。 感染NPEV后的疾病。尽管小鼠模型已被广泛用于深入了解 肠道病毒感染过程，人类和啮齿动物之间的遗传和生理差异限制了它们的 平移势此外，这些人的宿主因子相互作用中的物种不相容性 肠道病毒需要人受体、小鼠适应株或新生儿感染的过表达。 在为这项提议奠定基础的工作中，我们已经从多能干细胞中开发出了人类脊髓 脊髓类器官，重现了人类脊髓的一些细胞多样性。重要的是我们有 开创了一种将脊髓类器官中的运动神经元与人类骨骼连接起来的方法 肌肉和皮层神经元的制备，我们命名为神经胶质细胞。这些运动神经元形成 功能性神经肌肉接头，可以控制肌肉收缩。在这里，我们建议系统地 研究已知宿主因子在神经组织来源的细胞系中对EV-A71和EV-D 68的作用，发现 通过在神经细胞系中进行无偏基因组规模的遗传筛选， 谱系嗜性和肠病毒感染皮质-运动神经元样病变时对神经功能的影响。 我们的研究结果将揭示一组不同的关键受体和广泛作用的受体的作用和相对贡献。 宿主因素感染多种肠道病毒，发现并提供详细的分子机制， 神经细胞类型中的新宿主因子，并利用独特的神经类器官系统来揭示特定的 麻痹剂感染时对人体神经肌肉回路的趋向性和功能影响 肠道病毒EV-D 68和EV-A71。

PROJECT SUMMARY Enteroviruses are the leading cause of viral meningitis in children and recent outbreaks of emerging non-polio enteroviruses (NPEVs) have been associated with a polio-like paralysis named acute flaccid myelitis (AFM). Discovery and characterization of cellular components that are critical for neuropathogenesis hold promise for revealing new approaches to treat enterovirus disease. In recent years, multiple receptors have been identified for EV-A71 and EV-D68, NPEVs, which are most commonly associated with AFM. Using unbiased genome- scale screens, we have identified the phospholipase PLA2G16 as an entry factor acting immediately downstream of receptor engagement following NPEV infection. How the multiple receptors and PLA2G16 work together to enable infection in cell types relevant for neuropathogenesis is, however, largely unknown. Infection of cell types present in the central nervous system is critical for developing severe neurological forms of disease following infection with NPEVs. Although mouse models have been widely used to gain insights into enterovirus infection processes, genetic and physiological differences between human and rodents limit their translational potential. Moreover, species incompatibilities in host factor interactions of these human enteroviruses necessitate overexpression of human receptors, mouse-adapted strains or neonatal infections. In work that forms a foundation for this proposal, we have developed from pluripotent stem cells human spinal cord organoids that recapitulate some of the cell diversity of the human spinal cord. Importantly, we have pioneered an approach to functionally connect motor neurons in spinal cord organoids with human skeletal muscle and cortical neurons in a preparation we named assembloids. These motor assembloids form functional neuro-muscular junctions and can control muscle contraction. Here, we propose to systematically study the role of known host factors in cell lines derived from neural tissue on EV-A71 and EV-D68, discover novel host factors by performing unbiased genome-scale genetic screens in neural cell lines, and compare cell lineage tropism and effect on neuronal function during enterovirus infections of cortico-motor assembloids. Our results will reveal the role and relative contribution of a distinct set of critical receptors and broad-acting host factors to infection by multiple enteroviruses, discover and provide details on the molecular mechanism of novel host factors in neural cell types, and leverage a unique neural organoid system to uncover the specific tropism and functional effect on human neural-muscular circuits during infections with the paralytic enteroviruses EV-D68 and EV-A71.