Figure 4.

Knockout of USP22 impairs virus-triggered IRF3 nuclear translocation. (A and B) Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl BMDCs treated with 4-OHT (1 µM) for 3 d. The cells were infected with SeV, VSV, or HSV-1 for 4–8 h (A) or transfected with poly(I:C), ISD45, BDNA, HSV60, or HSV90 for 4 h (B) followed by qRT-PCR analysis of Ifnb, Isg15, Ccl5, and Usp22 mRNA. (C) ELISA analysis of IFN-β and CCL5 in the supernatants of Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl BMDCs treated as in A that were infected with VSV, HSV-1, or LPS for 12 h, or transfected with ligands for 6 h. (D) Immunoblot analysis of cytoplasmic and nuclear IRF3 in Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl BMDCs treated as in A that were infected with SeV, VSV, or HSV-1 for 0–8 h. (E) Immunofluorescence staining (with anti-IRF3) and microscopy imaging of BMDCs cells Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl BMDCs treated as in A that were infected with VSV or HSV-1 infection for 0–8 h. (F) Flow cytometry analysis (left graph) and microscopy imaging (right graph) of the replication of VSV-GFP or H129-G4 in Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl BMDCs treated as in A that were infected with VSV-GFP (multiplicity of infection [MOI] = 0.5) or H129-G4 (MOI = 1) for 1 h followed by PBS wash twice and cultured in full medium for 24 h. Numbers adjacent to the outlined areas indicate percentages of GFP⁺ BMDCs. (G) Plaque assay of the supernatants of Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl BMDCs treated as in A that were infected with VSV (MOI = 0.5) or HSV-1 (MOI = 1) for 1 h followed by PBS wash twice and cultured in full medium for 24 h. *, P < 0.05; **, P < 0.01; and ***, P < 0.001; n.s., not significant (two-way ANOVA followed by Bonferroni post-test or two-tailed t test). Scale bars represent 10 µm (yellow bars, E) or 200 µm (white bars, F). Data are representative of three (A–C) or two (D–G) independent experiments (graphs show mean ± SD, n = 3). SSC, side scatter.