Figure 10.

Reconstitution of KPNA2 into USP22 knockout cells restores virus-triggered IRF3 nuclear translocation. (A) qRT-PCR analysis of Ifnb, Ccl5, or Usp22 mRNA in Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl MEFs treated with 4-OHT (1 µM) for 3 d and reconstituted with empty vector (phage) or KPNA2 followed by infection with SeV or HSV-1 for 0–8 h. (B) Immunoblot analysis of cytoplasmic and nuclear IRF3 in Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl MEFs treated as in A followed by infection with SeV or HSV-1 for 0–8 h. (C) Microscopy imaging of the replication of GFP-VSV or H129-G4 in cells obtained in A infected with VSV-GFP (MOI = 0.5) or HSV-1-GFP (MOI = 0.5). (D) Plaque assay of the supernatants of Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl MEFs treated as in A followed by infection with VSV (MOI = 0.5) or HSV-1 (MOI = 0.5) for 1 h followed by twice PBS wash and cultured in full medium for 24 h. *, P < 0.05; ***, P < 0.001; n.s., not significant (two-way ANOVA followed by Bonferroni post-test). Scale bars represent 200 µm (C). Data are representative of three (A) or two (B–D) independent experiments (graphs show mean ± SD, n = 3).