A CRISPR-Cas9 screen reveals a role for WD repeat-containing protein 81 (WDR81) in the entry of late penetrating viruses

Abstract

Successful initiation of infection by many different viruses requires their uptake into the endosomal compartment. While some viruses exit this compartment early, others must reach the degradative, acidic environment of the late endosome. Mammalian orthoreovirus (reovirus) is one such late penetrating virus. To identify host factors that are important for reovirus infection, we performed a CRISPR-Cas9 knockout (KO) screen that targets over 20,000 genes in fibroblasts derived from the embryos of C57/BL6 mice. We identified seven genes (WDR81, WDR91, RAB7, CCZ1, CTSL, GNPTAB, and SLC35A1) that were required for the induction of cell death by reovirus. Notably, CRISPR-mediated KO of WD repeat-containing protein 81 (WDR81) rendered cells resistant to reovirus infection. Susceptibility to reovirus infection was restored by complementing KO cells with human WDR81. Although the absence of WDR81 did not affect viral attachment efficiency or uptake into the endosomal compartments for initial disassembly, it delayed viral gene expression and diminished infectious virus production. Consistent with the role of WDR81 in impacting the maturation of endosomes, WDR81-deficiency led to the accumulation of reovirus particles in dead-end compartments. Though WDR81 was dispensable for infection by VSV (vesicular stomatitis virus), which exits the endosomal system at an early stage, it was required for VSV-EBO GP (VSV that expresses the Ebolavirus glycoprotein), which must reach the late endosome to initiate infection. These results reveal a broad, previously unappreciated role for WDR81 in promoting the replication of late penetrating viruses.

Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis

“…Our findings parallel those of a study indicating a function for WD repeat-containing protein 81 (WDR81) in reovirus entry [69]. WDR81 was identified in a CRISPR/Cas9 cell-survival screen using mouse embryo fibroblasts and found to be required for a step in reovirus entry that follows ISVP formation.…”