The actin motor protein MYO10 facilitates post-entry spread of respiratory syncytial virus

Human respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants. However, host factors that influence disease severity remain incompletely defined. While clinical risk factors are known, identifying genetic susceptibility has been challenging. In this study, we combined human genetics with functional virology to identify host factors that modulate RSV infection and spread. Starting from a cohort of infants hospitalized with severe RSV disease, we prioritized rare coding variants present in homozygous form and predicted to cause strong functional impairment, and selected candidate genes for mechanistic follow-up. Functional interrogation of 23 candidates by CRISPR/Cas9 knockout screening in human lung epithelial cells identified unconventional myosin-X (MYO10), encoding the actin-based motor protein myosin-X, as a critical host factor for RSV. Genetic disruption or siRNA-mediated depletion of MYO10 significantly reduced RSV infectivity, with the strongest effects at post-entry stages of the viral life cycle. Loss of MYO10 impaired filopodia formation, cell migration, and wound healing, leading to altered cell–cell connectivity and restricted viral dissemination. MYO10 depletion reduced both short-range cell-to-cell transmission and longer-distance extracellular spread, resulting in fewer infected cells and diminished accumulation of progeny virus in culture supernatants. In contrast, RSV entry, early gene expression, and interferon responses were unaffected. Finally, a rare homozygous MYO10 motor-domain variant (rs7737765; H148Y), enriched in severe cases, also reduced RSV replication in cell culture—opposite to expectations for a risk allele—yet underscoring biological relevance and suggesting that MYO10 variation may influence disease in vivo through additional effects on epithelial function.