Glucosylceramide synthase maintains influenza virus entry and infection
Fig 2
CRISPR/Cas9 mediated knockout of glucosylceramide synthase.
(A) HEK 293 cells were pretreated with 20 μM PPMP (for 48 hours) or 100 nM bafilomycin (for 1 hour) and then infected with PR8 influenza virus encoding an NS1-GFP chimeric protein, in the presence of the indicated drug, for 18–24 hours (selected time points chosen after optimization). Cells were then lifted, fixed, and analyzed by flow cytometry for GFP expression. PPMP-treated samples exhibited a 50% reduction in GFP signal compared to WT, indicating a role for UGCG in influenza virus infection. Data represent the mean values of 4 biological replicates (each performed in triplicate) ± SE. (B) HEK 293 and A549 cells were transfected with plasmids encoding GFP as well as Cas9-sgRNA targeting UGCG. Cells were selected for GFP expression and single cell colonies were expanded and monitored for UGCG knockout as described in the Methods. (C,D) Selected cell clones (see S1 Fig) were assayed for UGCG activity by incubating cells with 5 μM C6-ceramide nanoliposome for 4 hours. Cells containing functional UGCG are able to convert C6-ceramide to C6-GlcCer, as seen in WT samples. HEK 293 and A549 UGCG KO cells displayed no C6-GlcCer, indicating a complete loss of UGCG activity. (E,F) Lipids from WT and KO cells were analyzed by mass spectrometry. In agreement with the measured enzyme activity (C,D), levels of total basal endogenous GlcCer were significantly reduced in both HEK 293 and A549 KO cells as compared to WT. Data represent the mean ± SE (n = 6 samples). ** p<0.01 using a Mann-Whitney non-parametric test.