Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense

Host interferons (IFNs) powerfully restrict viruses through the action of several hundred IFN-stimulated gene (ISG) products, many of which remain uncharacterized. Here, using RNAi screening, we identify several ISG restriction factors with previously undescribed contributions to IFN-mediated defense. Notably, RABGAP1L, a Tre2/Bub2/Cdc16 (TBC)-domain-containing protein involved in regulation of small membrane-bound GTPases, robustly potentiates IFN action against influenza A viruses (IAVs). Functional studies reveal that the catalytically active TBC domain of RABGAP1L promotes antiviral activity, and the RABGAP1L proximal interactome uncovered its association with proteins involved in endosomal sorting, maturation, and trafficking. In this regard, RABGAP1L overexpression is sufficient to disrupt endosomal function during IAV infection and restricts an early post-attachment, but pre-fusion, stage of IAV cell entry. Other RNA viruses that enter cells primarily via endocytosis are also impaired by RABGAP1L, while entry promiscuous SARS-CoV-2 is resistant. Our data highlight virus endocytosis as a key target for host defenses. [Display omitted] •An siRNA screen identifies genes contributing to the antiviral action of interferon•RABGAP1L potentiates the action of interferon against multiple RNA viruses•The RABGAP1L proximal interactome reveals its association with endosomal proteins•RABGAP1L disrupts endocytosis to limit cell entry of viruses using this pathway Interferons inhibit viruses through the concerted action of many host restriction factors. Here, Fernbach et al. use siRNA screening to identify interferon-dependent host factors that limit early stages of the influenza A virus replication cycle, leading to the characterization of RABGAP1L-mediated disruption of endocytosis as a broadly acting host antiviral mechanism.