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

项目总结 肠道病毒是儿童病毒性脑膜炎和最近新出现的非脊髓灰质炎暴发的主要原因 肠道病毒(NPEV)与一种名为急性弛缓性脊髓炎(AFM)的脊髓灰质炎样麻痹有关。 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疾病的新方法。近年来，多种受体已被鉴定出来。 对于EV-A71和EV-D68，NPEV是最常见的AFM相关病毒。利用无偏见的基因组- 规模筛选，我们已经确定磷脂酶PLA2G16是一个立即起作用的进入因子 NPEV感染后受体结合的下游。多受体和PLA2G16的作用机制 然而，与神经发病相关的细胞类型中的共同感染在很大程度上是未知的。感染 中枢神经系统中存在的细胞类型对于发展严重的神经性形式的 感染非传染性非典型肺炎病毒后的疾病。尽管老鼠模型已被广泛用于了解 肠道病毒的感染过程，人和啮齿动物之间的遗传和生理差异限制了他们的 翻译潜力。此外，这些人在宿主因素相互作用中的物种不亲和性 肠道病毒需要人类受体的过度表达、小鼠适应的菌株或新生儿感染。 在为这一提议奠定基础的工作中，我们从多能干细胞培育出了人类脊柱 脊髓有机体，概括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进行功能性连接的方法 肌肉和大脑皮层神经元，我们将其命名为集合体。这些马达装配体形成 有功能的神经-肌肉连接，可以控制肌肉收缩。在这里，我们建议系统地 研究已知宿主因素在EV-A71和EV-D68上的作用，发现神经组织来源的细胞系 通过在神经细胞系中进行无偏见的基因组规模的遗传筛选，并比较细胞 肠道病毒感染皮质运动集合体时的谱系趋向性及其对神经功能的影响。 我们的结果将揭示一组不同的关键受体和广泛作用的作用和相对贡献 宿主因素对多种肠道病毒感染的影响，发现并提供关于肠道病毒感染的分子机制的详细信息 神经细胞类型中的新宿主因子，并利用独特的神经器官系统来揭示特定的 瘫痪患者感染时的趋向性及其对人体神经肌肉环路的功能影响 肠道病毒EV-D68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.