Abstract
Viruses rely on egress machinery to exit cells, a step that is crucial for their transmission. While non-enveloped RNA viruses, such as enteroviruses, are typically associated with the lytic release pathway, emerging evidence suggests they can also be packaged within vesicles for non-lytic release. However, the molecular mechanisms that regulate viral release remain unclear. Here, we performed a genome-wide CRISPR-Cas9 screen in A549 cells and identified the microRNA hsa-miR-3156-1 as a critical host factor for enterovirus infection. Both the primary and mature forms of miR-3156-1 were upregulated following infection, with miR-3156-3p acting as a positive regulator of viral infection by directly targeting the 3'-untranslated regions of the K+ channel-associated genes KCNV1, KCNJ2, KCNK3, and KCNK5, thereby causing plasma membrane depolarization and Ca2+ influx, which increase viral release. Pre-administration of a miR-3156-3p antagomir alleviated virus-associated acute flaccid paralysis in an ICR neonatal mouse model. Our findings reveal how the miR-3156-3p/K+ channel axis controls enterovirus release, highlighting potential prophylactic targets.