Fatty acid 2-hydroxylase facilitates rotavirus uncoating and endosomal escape

Despite the clinical significance of many nonenveloped viruses, the molecular mechanisms of their internalization and membrane penetration are not well understood. Rotaviruses (RVs) are nonenveloped double-stranded RNA viruses and the leading cause of severe dehydrating diarrhea in infants and young children. We identified fatty acid 2-hydroxylase (encoded by <i>FA2H</i>) in the fatty acid 2-hydroxylation pathway as a proviral gene that supports RV infection. Genetic ablation of <i>FA2H</i> interfered with an early step in RV entry for multiple human and animal strains. Intestinal epithelial cell-specific deletion of <i>Fa2h</i> limited RV replication and diarrhea incidence in vivo. Using transmission electron microscopy and immunofluorescence, we found that viral particles were trapped in early and late endosomes in <i>FA2H</i> knockout cells, preventing their further exit into the cytosol. The defect in RV infectivity could be partially restored by treatment of cells with long-chain 2-hydroxy ceramides or a calcium channel activator that promotes Ca²⁺ efflux from endosomes. Both Junín virus, an arenavirus, and Shiga toxin, dependent on endosomal Ca²⁺ transport, required FA2H for efficient entry. Together, this study highlights a role of fatty acid 2-hydroxylation in RV entry into host cells and implicates 2-hydroxy ceramides as potential key regulators of endosomal Ca²⁺ levels, offering important insights for the development of host-directed therapies targeting fatty acid 2-hydroxylation to control microbial infections.