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 reduced 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 previously unappreciated role for WDR81 in promoting the replication of viruses that transit through late endosomes.

A Genome-wide CRISPR screen unveils the endosomal maturation protein WDR91 as a promoter of productive ASO activity in melanoma

“…This protein represents a strong candidate as ASO activity enhancer and shows that ASOs productive release in cytosol could mainly occur during early endosome maturation or from the late endosomes as supported by other studies 55,56 . Furthermore, these data support other studies highlighting WDR proteins 81 and 91 as important mediators of release and efficacy of internalized reoviruses or antibody-drug conjugates in cells during the early endosome maturation or from the late endosomes 57,58 .…”

Cholesterol-Dependent Membrane Deformation by Metastable Viral Capsids Facilitates Entry

“…Endosomal proteases disassemble particles to form ISVPs. Based on the evidence that ISVP to ISVP conversion is needed to cross the membrane, we expect that ISVPs and ISVP*s exist within endolysosome compartments [22] . In the experiments, we transiently transfected Vero 76 cells with a lysosomal-associated membrane protein 1 (LAMP1) GFP construct and then infected cells with reovirus (T1L/T3DM2).…”

HEATR5B associates with dynein‐dynactin and promotes motility of AP1‐bound endosomal membranes

“…These factors are candidates to participate in dynein‐mediated trafficking of messenger ribonucleoprotein particles (mRNPs) (Mofatteh & Bullock, 2017). Other intriguing hits include the dynactin‐stimulated tail interactor Wdr91, a Rab7 effector implicated in endosomal recycling and lysosomal function (Xing et al , 2021; Liu et al , 2022), as well as reovirus infection (Snyder et al , 2022). In addition to analysing the current set of dynein interactors, it may be possible in the future to adapt our pulldown approach to identify more transient interactors of the motor complex, for example by incorporating a biotin ligase on the dynein tail for proximity‐dependent biotinylation (Samavarchi‐Tehrani et al , 2020).…”