Glucosylceramide Maintains Influenza Infection through Regulation of Cellular Endocytosis

Influenza is an enveloped negative sense RNA virus encapsulated in a lipid bilayer derived from the host cell plasma membrane. Lipids play diverse and crucial roles for many viruses, including influenza. Previous studies have shown that influenza infection depends on cellular lipids, including the sphingolipids sphingomyelin and sphingosine. Here we examined the role of a third sphingolipid, glucosylceramide, in influenza infection following CRISPR/Cas9-mediated knockout of its metabolizing enzymes: glucosylceramide synthase (UGCG) and glucosylceramidase (GBA). We first confirmed the knockouts (in HEK 293 and A549 cells) by both western blotting and lipid mass spectrometry. We next observed diminished influenza infection in all four KO cell lines using a PR8 GFP reporter virus. We further showed that the reduction in infection correlates with impaired virus entry, using βeta-lactamase reporter particles. To examine whether glucosylceramide homeostasis is similarly required for other viruses, we compared entry mediated by the glycoproteins of influenza, VSV, Ebola, and Measles in GBA and UGCG knockout cells. Among these, GBA and UCGC loss significantly inhibited entry of particles bearing the glycoproteins of both influenza and Ebola, viruses that enter the cytoplasm through late endosomes. Consistent with the defect in late endosomal virus entry, we found that influenza particles in GBA knockout cells were impaired in trafficking to late endosomes (via colocalization with Lamp1). As an extension, we found that trafficking of epidermal growth factor to late endosomes as well as degradation of epidermal growth factor receptor were impaired in GBA knockout cells. Collectively our findings suggest that glucosylceramide is critically important for normal endocytic trafficking of viruses such as influenza as well as cellular cargos including growth factor receptors. In the viral context, modulation of glucosylceramide levels may represent a novel accompaniment to strategies to antagonize ‘late penetrating’ viruses, including influenza.