Enterovirus D68 2A protease causes nuclear pore complex dysfunction and motor neuron toxicity

The picornavirus Enterovirus D68 (EV-D68) is an important pathogen associated with acute flaccid myelitis (AFM). The pathogenesis of AFM involves infection of spinal motor neurons and motor neuron death, however the mechanisms linking EV-D68 infection to selective neurotoxicity are not well understood. Dysfunction of the nuclear pore complex (NPC) has been implicated in motor neuron injury in neurodegenerative diseases such as amyotrophic lateral sclerosis, and the NPC is also modified by picornavirus proteases during the course of infection. We therefore sought to determine the impact of EV-D68 proteases on NPC composition and function and their role in motor neuron toxicity. We demonstrate widespread disruption of NPC composition by EV-D68 2A and 3C proteases via the direct cleavage of a relatively small number of nucleoporins, notably Nup98 and POM121 by 2A^pro. Using reporter systems, we demonstrate that 2A^pro inhibits nuclear import and export of protein cargoes and also disrupts the permeability barrier of the NPC, while having no apparent effect on RNA export. We further show that 2A^pro is toxic to induced pluripotent stem cell derived motor neurons by demonstrating a rescue of toxicity with 2A^pro inhibitor telaprevir at concentrations that are insufficient to inhibit viral replication. This study expands our understanding of EV-D68 neuropathogenesis and provides a rationale for targeting the NPC or 2A^pro therapeutically in AFM.