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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 infection. We therefore sought to determine the impact of EV-D68 proteases on NPC composition and function. We demonstrate widespread disruption of NPC composition by EV-D68 2A and 3C proteases via direct cleavage of a relatively small number of nucleoporins, notably Nup98 and POM121, by 2A\n                    <jats:sup>pro<\/jats:sup>\n                    . Using reporter systems, we demonstrate that 2A\n                    <jats:sup>pro<\/jats:sup>\n                    inhibits nuclear transport of protein cargoes and disrupts the permeability barrier of the NPC, while having no apparent effect on RNA export. Independently, we show 2A\n                    <jats:sup>pro<\/jats:sup>\n                    is toxic to induced pluripotent stem cell-derived motor neurons by demonstrating a rescue of toxicity with the 2A\n                    <jats:sup>pro<\/jats:sup>\n                    inhibitor telaprevir at concentrations insufficient to inhibit viral replication. These findings expand our understanding of EV-D68 neuropathogenesis and provide a rationale for studying the NPC or 2A\n                    <jats:sup>pro<\/jats:sup>\n                    as therapeutic targets in AFM.\n                  <\/jats:p>","DOI":"10.7554\/elife.108672","type":"journal-article","created":{"date-parts":[[2025,9,29]],"date-time":"2025-09-29T14:31:30Z","timestamp":1759156290000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.108672","source":"Crossref","is-referenced-by-count":1,"title":["Enterovirus D68 2A protease causes nuclear pore complex dysfunction and independently contributes to motor neuron toxicity"],"prefix":"10.7554","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-6370-6858","authenticated-orcid":true,"given":"Katrina M","family":"Zinn","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05q6tgt32","id-type":"ROR","asserted-by":"publisher"}],"name":"Hugo W. Moser Research Institute, Michael V. 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