Fig. 1. LYSET is a critical host factor for multiple viruses that utilize activated cathepsins for entry.

(A) CRISPR screen for reovirus T3D (ReoT3D) host factors in U87MG cells. Significance of enrichment was determined via MAGeCK analysis (y-axis). All genes are plotted as bubbles on the x-axis, each representing a specific gene. A subset is color-coded according to function, labels show gene names. (B) Crystal violet staining after infection with ReoT3D representative of n=3 biologically independent replicates. “mock”: non-infected controls. (C) RT-qPCR quantification of ReoT3D RNA in infected U87MG and 293FT cells at MOI 1 at 72 hpi (mean ± SEM, n= 3) (D) U87MG or 293FT cells were infected with MOI of 1 ReoT3D virus. At 72 hpi, cells were lysed and viral titers determined via plaque assay (mean ± SEM, n = 3, ***p<0.001, ****p<0.0001; significance determined via one-way ANOVA with post-hoc Dunnett’s multiple comparisons test for (C) and (D)). (E) Time course of VSV-EBOV-GP infection of 293FT cells wild-type and LYSET KO (mean ± SEM, n = 3). (F) Bar-graph depicting independent infections of A549-ACE2 cells ± LYSET KO with SARS-CoV-2-mNeon using flow cytometry (mean ± SD, n = 3, ****p<0.0001; significance determined via unpaired, two-tailed student’s t test). (G) Infection of SARS-CoV-2 Delta and Omicron Spike reporter virus particles in Huh7.5.1 cell lines with or without LYSET KO. Cells were engineered to stably express ACE2 or ACE2 in combination with TMPRSS2, as indicated. Luciferase activity was measured at 72 hpi and normalized to wild-type cells (mean ± SEM, n = 6, ***p<0.001, ****p<0.0001; significance determined via significance was determined by unpaired, two-tailed student’s t-test with Welch’s correction).