Glycosphingolipids are essential entry factors for non-influenza orthomyxoviruses.

Thogotoviruses and quaranjaviruses are arthropod-borne orthomyxoviruses that circulate widely in wildlife and domestic animals and include several zoonotic members. Despite their close phylogenetic relationship to influenza viruses, the mechanisms underlying their replication remain poorly understood, and the host factors mediating viral entry are unknown. Here, we performed genome-wide loss-of-function CRISPR-Cas9 screens using replication-competent recombinant vesicular stomatitis viruses expressing thogoto- and quaranjavirus glycoproteins to identify cellular determinants of viral entry. These screens identified the glycosphingolipid (GSL) biosynthesis pathway as a key regulator of viral entry, with the upstream enzyme uridine diphosphate (UDP)-glucose ceramide glucosyltransferase (UGCG) emerging as a central host entry factor. Pharmacological inhibition of UGCG impaired thogoto- and quaranjavirus entry. The importance of GSL biosynthesis for thogotovirus replication was further validated using multiple thogotovirus isolates. Together, our findings establish GSLs as critical host entry factors for non-influenza orthomyxoviruses and identify UGCG as a potential target for antiviral intervention.