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

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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.