Figure 2: WT ISG15 and ISG15ΔGG restore VSV infection phenotype in ISG15-deficient cells.

(a–c) hTert-immortalized fibroblasts from ISG15-deficient patients (n=3) or controls (n=2 or 3) were treated with the indicated concentration of IFN-α2b for 12 h, washed, and allowed to rest for 36 h before infection. (a) Cells were infected with VSV at an MOI of 1.0 for 24 h. Supernatants were titered by TCID₅₀. (b) Cells were infected with VSV–GFP at an MOI of 1.0 for 24 h, fixed, subjected to nuclear staining, and imaged. Representative images are shown for each treatment group. (c) Quantification of panel b. Shown is the percentage of cells positive for both GFP and nuclear staining. (d) and (e) hTert-immortalized fibroblasts from ISG15-deficient patients (n=3) or controls (n=2), untransduced or stably transduced with luciferase, ISG15 or ISG15ΔGG, were mock-treated or primed with 1,000 IU ml⁻¹ IFN-α2b for 12 h, washed, and allowed to rest for 36 h. (d) Relative IFIT1 mRNA levels 48 h post-priming. (e) Fibroblasts were infected with VSV at an MOI of 1.0, 48 h post-priming. Supernatants were collected at 24 h post-infection and titered by TCID₅₀ in duplicate. a,d and e show the combined results of three experiments. b and c show single representative experiments of three performed. Error bars, s.d. Comparisons made with unpaired t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. NS, not significant.