Fig. 2.

ISGylation and viral susceptibility in cell lines derived from patients with mutations in ISG15. Control SV-40-immortalized fibroblasts (C1), fibroblasts derived from P1, P2 or a STAT1^−/− patient were either left untreated or treated with IFN-β for 24 hours. Cell extracts were analyzed by SDS-PAGE and immunoblotting with antibodies against ISG15, IFIT3 or tubulin (A). SV-40 fibroblast cell lines from patients P1 and P2 were mock-transfected (M), transfected with a plasmid encoding 3XFLAG-ISG15 (WT) or transfected with a plasmid encoding a form of ISG15 unable to conjugate with proteins (AA). Eighteen hours after transfection, we treated the cells with IFN-β for an additional 18 hours. Cell extracts were analyzed by SDS-PAGE and immunoblotting with FLAG, IFIT3 or tubulin antibodies (B) (A and B are representative of at least 3 independent experiments ). HSV-1 replication was monitored by assessing the fluorescence of GFP fused to a viral capsid protein, in SV40-fibroblasts from a healthy control C1, P1, P2 and a STAT1^−/− patient, infected with HSV-1 at a multiplicity of infection (MOI) of 0.2 for the times indicated. Cells were treated with either medium alone (C) or with IFN-α (D) for 24 h before infection. The results shown are the means of four independent experiments. Error bars indicate the SEM.